대한정형외과연구학회지제 9 권제 1 호 J. of Korean Orthopaedic Research Society Volume 9, Number 1, April, 2006 섬유소와혼합된자가골모세포주입에의한토끼장관골골결손의치료 가톨릭대학교의과대학정형외과학교실, 세원셀론텍 * 이승구 김석중 장정호 * 장재덕 * 박현신 * 박종수 = Abstract = Treatment of Long Tubular Bone Defect of Rabbit Using Autologous Cultured Osteoblasts Mixed with Fibrin Seung-Koo Lee, M.D.,Seok-Jung Kim, M.D., Cheong-Ho Chang, M.D.*, Jae-Deog Jang, Ph.D.*, Hyun-Shin Park,C.D.*, Jong-Soo Park, M.D. Department of Orthopedic Surgery, College of Medicine, The Catholic University of Korea, Central research institute, Cellontech*, Seoul, Korea Purpose: The osteogenic potential of autologous cultured osteoblasts mixed with fibrin when transplanted to bone defects was evaluated. Materals and Methods: Radial shaft defects over 15 mm were made in 30 New Zealand white rabbits. Fifteen rabbits in the control group underwent an iliac bone graft and 15 rabbits in the experimental group underwent an autologous cultured osteoblast injection mixed with fibrin. Both groups were compared radiologically and 5 rabbits in each group were sacrificed for histological evaluation using H-E and Masson s trichrome stains at 3, 6, and 9 weeks. Results: Osteogenesis in the control group progressed more rapidly than in the experimental group. However, at 9 weeks, bone formation in both groups were similar and showed no significant difference in terms of the amount of bone formation and the quality of bone union. Conclusion: Autologous cultured osteoblast transplantation mixed with fibrin in bone defects was found to produce bone efficiently. Key Words: Osteoblast, Fibrin, Osteogenesis, Autologous transplantation 통신저자 : 김석중경기도의정부시금오동 65-1 가톨릭대학교의정부성모병원정형외과 TEL: 031) 820-3066 FAX: 031) 847-3671 E-mail: peter@catholic.ac.kr 본논문의요지는보건복지부신약개발지원사업 (0405-DB00-0101-0007) 과제의연구비로이루어졌슴. 29
대한정형외과연구학회지제 9 권제 1 호 2006 년 서 론 대상및방법 외상, 종양의수술, 골질환등으로인하여사지골의일부분을잃게되는경우그환자의여생에있어서의장해는대단히커서, 정상적인골격계의기능을재건하고자하는많은노력이시행되어왔지만많은제한점과문제점들을가지고있다. 배양된자가골모세포를골이식대신에사용하는경우에는, 일반적인자가골이식시생길수있는공여부위의문제점 1) 과동종골이식시생길수있는면역학적문제점, 질병의전파와같은문제점들 22) 이해결될수있으며, 빠른회복을도모할수있다. 실험적으로골수기질세포 (bone marrow stromal cell) 는주변조직환경에따라골모세포, 연골모세포, 섬유모세포또는지방세포등으로분화할수있는능력을갖고있다고알려져있다 5,19). 그러나골수기질세포는골수내수효가극히적어 6), 이를실험에이용하기위해서는세포배양이필수적이며, 이러한잠재적분화능력을가진골수기질세포가골절부위의유합에도움을줄것이라고예상할수있다 20). 그러나세포만을이식할경우골결손이있는위치에머물지못하는임상적한계가있을수있다. 세포를이용하여골형성을촉진하고자, 합성물질과함께배양하여이를골결손부위에이식하고자하는시도가있으나 7) 이식을위해다시절개를가해야하고추가로감염이될수있는단점을가지고있다. 만일골모세포가주사기를이용하여원하는부위에주입이가능하다면골형성을효과적으로이룰수있다. 따라서생체내에서쉽게흡수되고, 최근성장인자를전달하기위해매개체로체로사용되고있는섬유소 (fibrin) 를이용하여 12) 이식된세포가이식부위에머물러효과적인골형성을할수있도록도와준다면, 주입형뼈세포치료제를만들수있는구체적인방법이될수있다. 본연구는토끼의골수기질세포를배양과정에서골모세포로분화시켜이를섬유소와혼합하여토끼의골결손부위에주사기를이용하여자가이식한후이식된골모세포의골형성능과조직학적특성에대해알아보고, 그실험결과를임상적으로적용할수있는가능성을찾고자하였다. 1. 실험동물실험동물로는암, 수구별없이체중약 3 kg내외의 New Zealand 백색토끼 30 마리를실험일주일전부터동일사료및동일조건하의사육장에서사육하였다. 이중 15마리는자가골모세포이식을위한실험군으로하였고, 나머지 15 마리는자가골이식을위한대조군으로하였다. 2. 골수기질세포의채취및골모세포배양토끼의후장골능에서채취한골수가포함된골편을 30 ml의 10% FBS -αmem 배양액 (Sigma Chemical Company, St. Louis, MO, USA) 과 350 unit의 heparin이든용기에넣어실험실로옮겼다. 이를조심스럽게흔들어섞은후큰골편은제거하고, 4 에서 472 g 로 10 분간원심분리를하였고, 골편은 collagenase(sigma) 를 24시간처리한후원심분리를하여상층액을제거하고남은침전물에 20 ml의배양액을첨가하였다. 여과기 (Falcon, Franklin lakes, NJ, USA) 를이용하여여과시킨후이를 T-75 조직배양플라스크 (Corning Science Products, Corning, NY, USA) 에 10 ml씩담아서배양을시작하였으며 16) 배양기 (Automatic CO 2 Incubator, Forma Scientific Inc, Marietta, Ohio, USA) 는 37, 5% CO 2 환경으로유지하였다. 다음날 dexamethasone 10~7 M(Sigma) 과 L-ascorbic acid(sigma) 50 μg/10 ml을첨가하고, 이후 3일마다 L- ascorbic acid를첨가하여세포들이골모세포로분화토록하였다. 광학현미경으로세포배양정도를평가하면서, 배양 5일째에배양액을교환하여주었고, 이후부터는 3일마다배양액교환후에 L-ascorbic acid를첨가하였다. 배양 14일째 alkaline phosphatase활성을확인하는 NBT- BCIP (nitro blue tetrazolium chloride (NBT) - 5-bromo-4-chloro-3-indolyl phosphate(bcip)) 염색을시행하고배양 4주무렵세포기질내에새로이형성된칼슘을확인하는 30
이승구외 섬유소와혼합된자가골모세포주입에의한토끼장관골골결손의치료 Alizarin red 염색을하여골모세포들이주로배양되었음을확인한후사용하였다. 배양 20 일째에배양액을제거한후 0.02% trypsin- ETDA(Gibco BRL, Gettysburg, PA, USA) 5 ml로씻어냈으며다시 0.02% trypsin- ETDA 3 ml를첨가하여배양기에 5분간두었다. 배양액 3 ml을가하여 trypsin-etda의활성을멈추게한뒤배양용기내의모든내용물을원추용기에모았으며, 이를 4 에서 265 g로 6 분간원심분리를한후, 상층액을제거하고나머지를모아세포수가 5 10 6 /ml가되게한후이를이식에사용하였다. 3. 골결손부위형성토끼를 ketamin 마취하에복와위로놓은후토끼의전완부를후방도달법에따라종절개하여요골간부를노출시켰다. 이후줄톱을이용하여길이 15 mm의골결손을만들었으며, 골결손부위의골막은철저히제거하였다 (Fig. 1A). 시술 후조심스럽게피부및피하조직을봉합하였다. 4. 골모세포및자가골이식대조군은형성된골결손부위에토끼의장골에서채취한해면골을이식하였으며, 실험군은섬유소 ( 그린플라스트, 녹십자, 한국 ) 와세포및배양액을 1:1 의비율로혼합하여이식하였다. 동물들은 gentamicin( 겐타마이신, 국제약품, 한국 ) (40 mg/day) 을이분하여근육주사하였고, 사육실에서자유행동을허용하였다. 5. 관찰방법실험후 3주, 6주및 9주에방사선촬영을시행하였으며, 최종방사선촬영에서골결손부의골형성의정도에따라결손부위를완전히채우는경우는 3점, 결손부위절반이상을채우는경우는 2점, 절반이하를채우는경우는 1점, 전혀골형성이되지않는경우는 0점으로하여실험의효과를평가 Fig. 1. The radiographs of 3 different rabbits in the control group at 3 weeks after the iliac bone graft. It shows good callus formation covering the defect(a, B, C). The radiographs of 3 different rabbits in the experimental group at 3 weeks after the osteoblast injection show callus formation in the defect(a, b, c). 31
대한정형외과연구학회지제 9 권제 1 호 2006 년 하였다 4). 각각의시기에경추탈구로대조군과실험군각각 5마리씩의토끼를희생시켰으며, 이식부위와골결손상부및하부의일부를함께적출하여 10% formalin에고정한후, 20% 수용성 formic acid와 10% sodium citrate 혼합액으로 3~4 일간탈석회화시키고 paraffin에포매하여약 6 μm 두께의조직표본을만들었다. 이를 hematoxylin-eosin( 이하 H-E) 염색및 Masson s trichrome 염색을시행하여조직을관찰하였다. 결과 1. 육안적관찰이식부위의육안적검사상 3, 6, 9주각시기의대조군과실험군모두비교적안정성을갖는단단한조직으로대치되었으며, 주변의골조직과분리되지않고연속적으로연결된양상을보였다. 2. 방사선학적관찰 1) 3주 : 대조군의경우이식부위에비교적강한골음영소견이보이고있으며, 골결손상부및하부, 이식부위사이에는경계가보이고있으나골결손상부와하부주변은강한골막반응과함께이식부위와연결성이형성되어가고있는양상을보였다 (Fig. 1A, B, C). 실험군의 3주의경우, 대조군에비해이식부위의골음영의정도와골막반응의정도가작고, 부분적으로결손부위가채워지지않은양상을보였으나, 이식부위와골결손상부및하부와의연속성은형성되었다 (Fig. 1a, b, c). 2) 6주 : 대조군의경우, 3주에비해형성된골의재형성이진행되어 3주에서미성숙해보였던가골음영은주변의정상골의음영과비슷한성숙골로재형성되는양상을보였다 (Fig. 2A, B, C). 실험군의경우, 3주에서약하게보였던골음영은좀더강하게나타났으며, 3주방사선검사상결손부위처럼보였던부분은새로운골형성음 Fig. 2. The radiographs of 3 different rabbits in the control group at 6 weeks after the iliac bone graft show bone formation. The rabbits pictured are the same rabbits shown in Fig. 3(A, B, C). The radiographs of 3 different rabbits in the experimental group at 6 weeks after the osteoblast injection show bone formation in the defect. The rabbits pictured are the same rabbits shown in Fig. 3(a, b, c). 32
이승구외 섬유소와혼합된자가골모세포주입에의한토끼장관골골결손의치료 영을보였다 (Fig. 2a, b, c). 3) 9주 : 대조군과실험군에서이식부분의골음영형성소견과함께주변골과특정한경계부위없이연속성이생기고, 일부에서는완전한골수강이형성되어방사선학적으로비교적완전히성숙한골의재형성소견을보였다. 방사선학적골형성수치상 9주의실험군과대조군모두에서 3점으로골결손상부및하부와의골유합결과를보였다 (Fig. 3A, B, C)(Fig. 3a, b, c). 3. 조직학적관찰 1) 3주 : 실험군 3주의섬유소와혼합되어골모세포가이식된골결손부위는독립적으로골형성을보이는조직 (Fig. 4a, b) 과연골형성을보이는조직 (Fig. 4A, B) 이혼재되어있었으며, 방사선상빈공간으로보였던부분도이러한조직으로채워져서골형성에참여하고있었다 (Fig. 5A, B). 또한새로이형성되어가는골조직의주 변으로활발한아교질형성과이것의주변조직으로의연계양상이관찰되었으며일정치는않지만아교질의형성에방향성이관찰되었다 (Fig. 6). 이와함께실험군에서는미성숙상태의혈행형성과연골내골화, 미성숙층판골의형성이이식부위를중심으로관찰되었으며, 대조군 3주의경우는실험군에비해좀더성숙한조직의형성이관찰되었는데, 골막및피질골이형성되어골결손상부및하부의주변과연결되고있었으며, 내부는이식골이흡수되고, 골수강이형성되어주변의정상골과연결되는양상이관찰되었다 (Fig. 5a, b). 2) 6주 : 실험군 6주에서는 3주에서의연골형성을보이는조직이거의관찰되지않고미성숙층판골과성숙층판골의형성이현저하였으며, 대조군의경우성숙한골소주의형성과주변에비해크기가큰혈행형성이주로관찰되었다. 3) 9주 : 전반적으로시간에따른골조직의형성과성숙의속도는대조군에서빨리관찰되었으며, 9주에서는실험군에서도성숙골이형성되어주변 Fig. 3. The radiographs of 3 different rabbits in the control group at 9 weeks after the iliac bone graft show bone formation. The rabbits pictured are the same rabbits shown in Fig. 3 and 4. Each shows good remodelling of the grafted bone(a, B, C). The radiographs of 3 different rabbits in the experimental group at 9 weeks after the osteoblast injection show bone formation in the defect. The rabbits pictured are the same rabbits shown in Fig. 3 and 4. Each figure shows a steady progression in remodelling(a, b, c). 33
대한정형외과연구학회지제 9 권제 1 호 2006 년 Fig. 4. H-E stain and Masson s trichrome stain of the black arrow area in Fig. 6 show chondrocyte metaplasia and bone formation( 100)(A, B). H-E stain and Masson s trichrome stain of the green arrow area from Fig. 6 shows immature lamellar bone and vascular formation ( 100)(a, b). Fig. 5. The radiograph of a experimental group rabbit at 3 weeks shows bone formation connecting both osteotomy sites. The defect is still not completely filled(a). The defect is completely filled with newly formed cartilagenous tissue(black arrow), lamellar bone tissue(blue arrow) and fibroosseous tissue(green arrow) in H-E stain of Fig. 6A.( 6)(B). The radiograph of a control group rabbit at 3 weeks shows bone formation connecting both osteotomy sites. (a). The defect shows continuity between both osteotomy sites by newly formed bone bridges in H-E stain of Fig. 6a.( 6)(b). 34
이승구외 섬유소와혼합된자가골모세포주입에의한토끼장관골골결손의치료 골조직과연결되는재형성의소견이관찰되었다. 9주의실험군과대조군모두에서혈행의증가와성숙된골소주가형성되어골결손부위는골조직으로대체되었다 (Fig. 7). 고찰대부분의의사들이선호하는골이식방법은자가골이식으로서, 동종골이식에비해훨씬좋은결과가보고되고있으나, 장골능에서골을채취하게되어술후통증이매우심하고, 입원기간과회복기간이길고, 치료비가많이들어가는단점이있다 1). 또한골이식의공여부가제한되어있고, 채취할수있는양도한정되어있는등의문제점이있다 10). 이러한단점을보완하기위해실시되어온동종골이식의경우주로사체에서골을얻어사용하게되는데골이식을위한추가적인이차수술이필요하지않다는장점을가지고있지만, 멸균과정에의한골강도의약화, 거부반응의발생, 간염이나 AIDS와같은전염성질환에감염이될 수있다는단점을가지고있다 13,22). 일반적인이식술외에이연구에서사용한자가골모세포이식의모태라할수있는골수주입술 (bone marrow injection) 은골수내의골생성전구세포 (osteoprogenitor cell) 가골형성을유도하고촉진한다고한주장에근거를둔다 2). 골수주입술은주로골절치유를위해단독으로주입되기도하지만, 골이식과병행하여주입되기도한다. 골수주입술은자가골이식술과는달리공여부위의피부절개를통한수술이아니므로공여부의문제점이없고, 합병증이나부작용이없다는것이큰장점이다. 그러나한곳에서뽑을수있는골수의양이제한되어있고골수에포함된골형성전구세포의수는매우제한되어있어있기때문에그효과또한확실치않다 17). 이런이유때문에세포를배양한후이식하는것이가장가능성이있는방법이라고생각하여배양세포를매개체에넣어서로다른종에이식한후성공적인결과를얻었다고보고하였다 5). 이연구에서섬유소를이용하여배양한골모세포를골결손부위에자가이식한 Fig. 6. Masson s trichrome stains of the blue arrow area from Fig. 6 show active collagen fiber formation and an increase in vascular ingrowth( 100)(A, B). H-E stain and Masson s trichrome stain of the blue arrow area from Fig. 6 show active collagen fiber and vascular formation. This formation is partially connected to the surrounding immature lamellar bone ( 100)(a, b). 35
대한정형외과연구학회지제 9 권제 1 호 2006 년 이유는이식된골모세포가결손부위내에안전하게부착할수있게함은물론섬유소는수술장에서지혈목적으로이용되는것으로안전하고이물반응없이쉽게흡수되는것으로알려져있기때문이다. 또한섬유소는성장인자를전달하는매개체가되어세포나성장인자만으로는이식물의부피를유지하기어려운곳에이식후주변세포의증식을유도하여, 섬유소가이식물의초기정착을위해중요한역할을할수있다 12). 그러나아직까지이식을위해사용되는세포의기원과형태학적특징들에대한정보나골형성과재형성을조절하는요인들에대한지식은제한되어있다. 그리고필요한세포수, 배양법, 시술법등에대한많은임상적인시도와연구를통해효과적인치료의방법에대한어느정도의공통된인식이필요한상태이다. Ashton 들 2) 과Bab 들 3) 도임상적사용을위한전단계로생체내 diffusion chamber를만들어골수기질세포가주변조직환경에따라골모세포, 연골모세포, 섬유세포로분화된다고보고하였 다. 이러한관점에서골수기질세포를배양했을때이들은주로골모세포쪽으로분화하게되며 8,9), 척추극돌기사이에이식하였을때골형성이일어남이보고되었다 15). 즉골조직이나그주변에배양한골모세포를이식하는경우에는골형성의가능성이높아져, 이를임상에도적용해볼수있다. 한편, 이연구에서는이식에필요한세포수를결정하는데있어기존의자료를통해가설을세웠는데, 새로형성되는골조직은해면골조직에가깝고이러한골조직에포함되는골세포의수는, 골세포가골조직에균등하게분포한다고가정하고 21) 부피단위로환산하면골 1 cm 3 당약4 10 5 개의골세포가존재하는것으로계산이된다. 따라서 5 10 6 개의골모세포가형성할수있는골의이론적인부피량은약 12.5 cm 3 이된다. 이중 50% 정도가재형성으로흡수된다 14) 하더라도, 나머지는 15 mm의크기의골결손부위를채우는충분한양이된다. 한편골결손부위의크기에대해서는, 토끼의요골간부에여러크기의골결손을만들어골형성이자연적으로일어나지않는골 Fig. 7. The radiograph of a experimental group rabbit at 9 weeks shows bone formation connecting both osteotomy sites. The defect is filled completely(a). The defect is filled completely with newly formed mature bone in H- E stain of Fig.9A.( 6)(B). The radiograph of a control group rabbit at 9 weeks shows complete bone formation and remodelling in the defect(a). The defect is filled with newly formed mature bone in H-E stain of Fig. 9a( 6)(b). 36
이승구외 섬유소와혼합된자가골모세포주입에의한토끼장관골골결손의치료 결손부위의크기 (critical size defect) 가 15 mm라는 Herold 들 11) 의연구에따랐으며, 이연구에서도같은크기의결손을만들어추시한결과골결손상부와하부가가늘어지고얇아져서더이상골형성이일어나지않음을관찰하였다. 따라서이이상크기의결손부위에골모세포를이식한후, 골형성을관찰하였기때문에, 이는이식한골모세포에의해서골형성이일어난것으로생각된다. 이연구의결과, 실험군 3주에서방사선학적으로는부분적인골형성양상이보였으나조직학적으로연골세포화생 (chondrocyte metaplasia), 독립된미성숙골과형성된새로운조직이결손부위를완전히채웠으며, 대조군에서는미성숙하지만방사선학적이나조직학적으로결손부위가골결손상부및하부와연결되어유합되는소견을보였다. 자가골을이식한골결손의중앙부는골조직이흡수되어골수강에해당하는공간이형성되었으며, 내부에는혈행형성과미성숙골조직관찰되었다 (Fig. 5). 대조군과실험군 9주에서는골결손부위를채우는완전한골화가관찰되었으며, 층판골의형성과혈행형성이더욱뚜렷해져골수강이서로연결되는재형성의소견을보였다 (Fig. 7). 실험군 3주의골형성은골결손상부및하부와중앙부에서모두관찰되었는데이는주변의혈종형성과이식된골모세포의작용에의한것으로여겨지는데, 특히골결손상부및하부와떨어진이식부에서도주변의골조직과연결성이없는독립된골형성이발견된것으로보아섬유소와혼합되어함께이식된골모세포가골결손부위내에서생존하고이것이골형성을유도한것으로생각된다 (Fig. 5). 이연구에서새로이형성된대부분의연골및골조직들이어느한곳에치우치지않고결손부위에서고르게위치하고있었는데, 이는섬유소가이식초기에골모세포들의위치를잡아주는매개체의기능을한것으로생각되며, 이외에도토끼는이식수술후통증과마취때문에움직임이적어골모세포가이식된부위에서자리를잡을수있는충분한시간을주었을가능성도있을것이다. 골모세포를 calcium phosphate ceramic과함께피하조직내이식을하여혈액공급이좋은주변부에선막내골화가, 그리고내부 에선연골내골화가일어남이관찰된바있고 18), diffusion chamber에이식된골수기질세포가골, 연골과함께제 1형및제2형의아교질을형성하는데이렇게서로다른형의아교질이같은세포내에서형성되는것은직접적인혈액순환의차단이하나의원인이라는보고가있다 3). 이실험의실험군 3주의결과에서도이식부위의중앙부에연골조직의형성이관찰되는데이또한수술로인해주변부로부터의혈액순환이원만하지못하게되어이식한골모세포가연골화과정을거쳐골형성의단계로서서히이행되는것으로판단된다. 그외이식초기에는비록골수기질세포를골모세포로분화시켜이식했다하더라도미분화과정에있을수있어, 이러한환경에따라연골세포로분화되었을가능성도있을것으로사료된다. 만일이방법이경피적으로사용될경우에는만족할만한골형성을얻을수있을것으로기대되는데그이유는이식시에조직손상이없어주변의혈액공급을차단하지않고, 이식된골모세포에의해형성된미성숙골조직이쉽게주변의조직과연계되기때문으로생각된다. 이연구에서전반적으로자가골이식과비교해서골모세포이식은골형성이나성숙의속도가늦고범위가작았지만골결손을치료하기위한골형성의면에서는큰차이를보이지않았으며, 초기방사선학적결과에비해조직학적으로는활발한골조직의형성소견을보였다. 또한최종추시결과에서는방사선학적및조직학적으로두군사이에차이를볼수없어골이식에못지않은결과를나타내었다. 골절의유합을위해서자가골이식은가장빠르고효과적인방법임에는틀림이없지만, 수술로인한공여부위의통증과이식골의양적인제한점, 그리고이식을위해추가의수술이필요하다는점들을고려한다면, 골유합의효과를낼수있는골모세포이식술은이러한자가골이식술의대안이될수있다고생각된다. 이연구결과를통해골모세포주입술이골질환이나외상으로인한골결손의치료에많은기여를할것으로기대한다. 결론골결손부위에섬유소와함께자가이식된골 37
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