Korean Journal of Oral and Maxillofacial Pathology 2015;39(2): ISSN: (Print); (Online) Available online at

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1 Korean Journal of Oral and Maxillofacial Pathology 2015;39(2): ISSN: (Print); (Online) Available online at 세포부착 PHBV- 교원질나노섬유의골유도재생효용성평가 신홍인 경북대학교치의학전문대학원구강병리학교실 <Abstract> The Evaluation of PHBV-Collagen Nonofibrous Membrane for Efficient Guided Bone Regeneration Hong-In Shin Department of Oral Pathology, School of Dentistry, Kyungpook National University Daegu, Korea This study was performed to improve the efficiency of guided bone regeneration by adapting the tissue-engineered bone regeneration approach. The PHBV-collagen nonofibrous membranes cultured with rat calvarial periosteum derived cells in osteogenic culture medium for 7 days were applied to 5 x 2mm size artificial rat tibial defects for 3weeks and evaluated its efficiency as an alternative cell engineered membrane for effective guided bone regeneration by routine histological observation. The defects covered with cell attached PHBV-collagen nanofibrous membrane complexes showed favorable bone repair compared to both membrane non-covered control and membrane only covered group without specific side effects. These findings suggest that the favorable guided bone generation can be achieved by adapting the tissue engineered bone regeneration approach using PHBV-Collagen nanofibrous membrane scaffold. Key words:phbv-collagen Nanofibrous Membrne, Guided Bone Regeneration, Tissue-Engineered Bone Regeneration Ⅰ. INTRODUCTION 최근각광을받고있는조직공학적재생술은지지체에줄기 세포또는특정조직을형성할수있는분화된세포, 또는특정 유전자및생활성물질을부착시켜재생이필요한부위에이식 함으로써보다효과적인재생을도모하고자하는술식이다 1-3). 효과적인조직공학적재생을이루기위해서는원하는부위 에서원하는조직을형성할수있는세포의효과적인도입이 관건이된다. 이는지지체에분화된세포를직접부착시켜 이식하거나또는재생이요구되는부위에존재하는미분화 세포를원하는세포로분화를유도할수있는유전자나생활 * Correspondence: Hong-In Shin Correspondence: Hong-In Shin, Department of Oral Pathology, School of Dentistry, Kyungpook National University Daegu, Korea. Tel: , Fax: hishin@knu.ac.kr Received: Mar 21, 2015; Revised: Mar 25, 2015; Accepted: Mar 28, 2015 성물질을효과적으로도입시킴으로써이룰수있다 4-5). 따라서재생효과의극대화를위해서는이런세포또는인자들을효과적으로도입하기위한지지체의개발과세포배양법의확립이선행되어야한다. 특별히골조직의재생을위해서는골의특성상하중을지지해야할필요가있을수있으므로골과유사한물리 화학적특성을살린지지체의개발이이어어져왔다 6-8). 다수의골재생용지지체는바이오세라믹과같은무기물을기반으로주로다공성구조의블록형태로제작되고있다 9). 그러나치조골에서와같이소량의골증대가요구되는경우에는과립또는분말형태의골이식체가널리활용된다. 치과용보철물의지지를위한치조골의증대가필요한경우골이식술과더불어골유도재생술이대표적인술식이되어왔다. 따라서세포가부착된막성지지체를차폐막형태로단독또는다양한과립형골이식제이식과병용함으로써효과 이논문은 2012 학년도경북대학교학술연구비에의하여연구되었음

2 적인조직공학적골재생을도모할수있을것으로기대된다. 본연구에서는생체적합성이양호한생분해성 PHBV(3- hydroxybutyrate-co-3-hydroxyvalerate) 를기본재료로하여교원질을 1:1 비율로혼합하여전기방사를통해직경 500nm 크기의나노섬유들이격자모양으로짜여진나노섬유막을제작하였다. 그리고여기에백서두개골막유래세포들을부착식켜백서경골골결손부에적용시켜조직공학적골유도재생술의효용성을평가하였다. Ⅱ. MATERIALS AND METHODS 1. 실험재료조직공학적골재생용기법을적용하여골유도재생술의효과를증대시키기위한세포지지용차폐막으로 PHBV와교원질을 1:1로섞어전기방사하여직경 500nm 크기의나노섬유들이무작위로격자모양으로짜여진 PHBV- 교원질나노섬유막을평가대상으로하였다. 실험동물은 200g 전후의 Sprague-Dawley 계웅성백서를활용하였다. 2. 실험방법가. 골결손부형성백서경골에인위적으로형성한골결손부의회복양상을평가하기위해먼저실험동물을마취한다음, 양쪽경골상방의털을면도칼로제거하고, 알콜용액으로소독하였다. 그리고 15번수술용칼로상방피부를 1cm 크기로절개하여경골을노출시킨후, 저속치과용엔진을이용하여발열이되지않도록생리식염수를수주하면서 5 2mm 크기의골결손부를형성하였다. 나. PHBV- 교원질나노섬유막및 PHBV 나노섬유막 -세포복합체이식 48well plate를이용하여백서두개골막유래의일차배양세포를 PHBV- 교원질나노섬유막에파종하여일주간골유도배양조건하에서배양한다음, 좌측에는세포를부착시키지 않은 PHBV- 교원질나노섬유막을우측에는세포를배양부착시킨 PHBV- 교원질나노섬유막을경골의골결손부에각각이식하였다. 그리고 5-0 vicryl 봉합사를이용하여층별봉합한후, 타도마이오셀 연고를도포하였다. 감염예방을위해추가적으로폭소린 (0.1cc/day) 을 3일간근주하였다. 섬유막을이식하지않은군을대조군으로하여동일한방법으로처리하였다. 다. 조직학적관찰실험 3주째실험동물을희생시켜양쪽경골을절취하여 10% 중성포르말린으로고정하였다. 통법에준하여 10% Formic acid 용액으로탈회시켜, 파라핀포매블록을제작한다음, 5um 두께의절편을형성하였다. 이들절편을 H&E 염색한후조직학적골성회복양상을광학현미경으로관찰하였다. Ⅲ. RESULTS 골결손부처치 3주째, 차폐막을위치시키지않은군에비해 PHBV-교원질나노섬유막만을위치시킨군에서보다양호한골성회복을이루었다. 그리고두개골막유래일차배양세포를 PHBV- 교원질나노섬유막에부착시켜골유도배양조건하에서일주간배양하여위치시킨실험군에서보다양호한골성회복을나타내었다. 차폐막을이식하지않은군에서는회복된피질골판이골수양결체조직이다수함유된불연속적인판상구조를보였고, 일부골조직들은골세포를다수함유한미성숙섬유골양상을나타내었다 (Figure 1A). 세포를부착시키지않는 PHBV-교원질나노섬유막을이식한군은비교적양호한판상구조의성숙된치밀골에의한회복을나타내었으나골수양결체조직이내부에함유되어완전한골판구조를이루지는못하였다 (Figure 1B). 반면, PHBV-교원질나노섬유막에백서두개골막유래세포를부착배양하여이식한군에서는조혈성골수영역이뚜렷이감소된보다치밀한층판골에의한양호한골성회복양상을나타내었다 (Figure 1C). 488

3 Figure 1. 술후 3 주째백서경골결손부의회복양상. A: 비이식대조군, B: PHBV- 교원질나노섬유막단독이식군, C: 백서두개골막유래배양세포부착 PHBV- 교원질나노섬유막이식군. (H-E, 원배율 40, 확대부 100) IV. DISCUSSION 일반적으로골조직은재생능력이양호한조직으로분류되며, 임계크기이하의골손상에서는골모세포들에의해반흔형성없이완전한골성회복을이룰수있다. 그러나여러원인에의해과도한골결손이이루어질경우는골이식술과같은부가적인술식이요구된다. 이런경우자가골이식을비롯한다양하게처리된동종골및이종골그리고생합성골이식체들에의한골성회복이다양한방법으로시도되고있다 10-12). 최근지지체에세포또는유전자및생활성물질등을부착시켜조직손상부위에이식하여효과적인재생을도모하는조직공학적재생술이확립되면서이들의재생효과를극대화하기위한다양한노력이경주되고있다 13). 치조골의경우다른부위의골조직과달리골결손부위가작은경우과립또는 분말형의골이식제를사용하게되므로이식된골이식제를효과적으로그위치에유지시키는것이용이하지않다. 따라서골유도재생술에활용되는다양한흡수또는비흡수성차폐막을이용하여골이식체를제위치시켜섬유조직의유입을차단하게된다. 최근에는이런차폐막대용으로자가혈에서얻어지는혈소판농축섬유소막 (PRF) 을이용함으로써골재생효과의극대화를도모하기도한다 14). 치조골의효과적인증대를위한또다른시도로차폐막에골형성을도모할수있는골모세포또는골손상부위의미분화세포들을골모세포로유도할수있는다양한생활성물질을부착시키는골유도재생술단독또는이들술식의골이식과병용을들수있다. 이에본실험에서는세포의부착보관이용이한 PHBV- 교원질나노섬유막을조직공학적골재생술을겸비한골유도재 489

4 생술의차폐막으로써의효용성을평가하였다. PHBV 는양호한생체적합성과흡수성을지닌물질로다양한물질과혼합하여전기방사를통해얻어지는직경 500nm 크기의섬유를격자모양으로무작위로짜여진얇은막으로형성할수있다. PHBV 에다양한성분들즉교원질또는젤라틴등과같은성분을함유시켜제작한 PHBV 기반나노섬유막에대한조직공학적지지체로서의유용성이여러실험에서긍정적으로평가되고있다. PHBV- 교원질나노섬유막의세포부착및증식능그리고냉동보관성의용이성과효용성이앞선실험에서확인되어본실험에서는이들세포- 지지체복합체의조직공학적골재생효과와골유도재생술의효과를평행시킴으로골성회복을극대화하기위한일환으로 PHBV- 교원질나노섬유막에백서두개골막에서채취한일차배양세포를골성유도배양조건하에서골모세포로의분화를유도하였다. 골유도배양조건에서일주간 PHBV- 교원질나노섬유막에부착시켜형성시킨세포 -PHBV- 교원질나노섬유막복합체를대안적차폐막으로하여인위적으로백서경골에형성한 5 2 mm 크기의골결손부에이식하였다. 그리고골막, 근육, 그리고피부순으로층별봉합하였다. 세포가부착된 PHBV- 교원질나노섬유막은큰어려움없이쉽게조작할수있었으며, 이들세포-지지체복합체는골결손부위에무리없이접합되었다. 그리고봉합사를이용하여골조직과틈이형성되지않도록긴밀히고정할수있었다. 시술 3주후골재생양상을조직학적으로비교평가한결과, 골결손부를막으로피개하지않은군보다 PHBV- 교원질나노섬유막을차폐막으로사용한군에서보다양호한골성회복을보였으며, 특히백서두개골막세포를부착배양하여이식한군에서더욱양호한골성회복양상을나타내었다. 이와같은소견은 PHBV- 교원질나노섬유막이골유도재생술의차폐막로서효과가인정될뿐아니라세포부착에의한조직공학적접근이골유도재생술의효과를상승적으로배가시킬수있음을시사한다. 백서의경우두피가얇아넓은두개골로부터쉽게두개골막을체취할수있고, 두개골막에존재하는세포들이골유도배양조건하에서양호한골성분화를이룬다는점에서이들세포들이조직공학적골재생술에좋은세포공급원이될수있을것으로평가된다. 그러나사람의 경우는두피가두꺼워상대적으로얇은피부조직을덮여있는경골의골막을활용하는것이효과적일수있다. 따라서 PHBV- 교원질나노섬유막을골유도재생술의차폐막으로활용하여상황에맞게골막또는골수, 치수, 지방등과같은다양한공급원으로부터세포를제공받아부착 배양시킴으로써세포-지지체복합체를형성하여통상적인골이식술및골유도재생술에조직공학적재생효과를부가시킴으로골재생효과를극대화할수있을것으로기대된다. Ⅵ. REFERENCES 1. Salgado AJ, Coutinho OP, Reis RL: Bone tissue engineering: state of the art and future trends. Macromol Biosci. 2004;4: Oryan A, Alidadi S, Moshiri A1, Maffulli N: Bone regenerative medicine: classic options, novel strategies, and future directions.j Orthop Surg Res ;9:18. doi: / X Cancedda R, Giannoni P, Mastrogiacomo M: A tissue engineering approach to bone repair in large animal models and in clinical practice, Biomaterials. 2007; 28: Hosseinkhani M, Mehrabani D, Manafi A, Shirazi R. Tissue engineered scaffolds in regenerative medicine. World J Plast Surg. 2014;3: Gothard D, Smith EL, Shakesheff KM, Oreffo RO: Tissue engineered bone using select growth factors: A comprehensive review of animal studies and clinical translation studies in man. Eur Cell Mater. 2014;28: Aichelmann-Reidy ME, Yukna RA: Bone replacement grafts. The bone substitutes. Dent Clin N Am 1998; 42: Babis GC, Soucacos PN: Bone scaffolds: the role of mechanical stability and instrumentation, Injury. 2005; 490

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00)14-1목차1~3 Effect of the bovine collagen matrix on bone formation in rat calvarial and mandibular defects Jeong-Ho Yun 1, Jee-Hee Jung 2, Jin-Woo Kim 2, Chang-Sung Kim 2, Seong-Ho Choi 2, Kyoo-Sung Cho 2 1 Department