연고형 Mineral Trioxide Aggregates (MTA) 와분말 - 용액혼합형 MTA 의골내이식시험비교연구 권영대, 석수황, 이상혁, 임범순 * 서울대학교치의학대학원치과생체재료과학교실 대한치과재료학회지 44(3) : 229-246, 2017 ISSN:2384-4434 (Print); 2384-3268 (Online) Available online at http://www.kadm.org http://dx.doi.org/10.14815/kjdm.2017.44.3.229 Comparison of intraosseous implantation between paste type mineral trioxide aggregates (MTA) and powder-liquid mix type MTA Young-Dae Kwon, Soohwang Seok, Sang-Hyeok Lee, Bum-Soon Lim * Dept. of Dental Biomaterials Science, School of Dentistry, Seoul National University, Seoul, Korea. The aim of this study was to evaluate the implantation of powder-liquid mix type MTA, paste type MTA, and resin-based endodontic sealer (AH Plus) in tibia of rabbits for 12 weeks. Intraosseous implantation was tested according to ISO 10993-6:2016. After anesthesia, the cortical bone of tibia was exposed, then three or two holes were formed (2 mm in diameter 6 mm in depth) in each side. The mixed materials according to manufacturer s instruction were placed into the cylindrical holes. After implantation, all animals received visual inspection at least once a week and inflammation, complication, and other specific figures were recorded. All animals were euthanized 12 weeks after implantation, and the specimens received visual and histologic evaluation, then following results were obtained. New bone formation were most-abundant and uniformly in the experimental group of implantation of paste type MTA, and were observed to some extent in the experimental group of implantation of powder-liquid mix type MTA and microvascular invasion around MTA was examined. As the control groups of implantation of epoxy resin based endodontic sealer, severe lymphocytes and neutrophils were observed. Histologic evaluation revealed that both paste type MTA and powder-liquid mix type MTA were non-irritant compared with AH Plus that had no potential risk biologically, and were seemed to meet the criteria of implantation test. As the paste type MTA has similar physical and biological properties with better handling behavior, so powder-liquid mix type MTA can be substituted by the paste type MTA. Key words: Mineral trioxide aggregates, Paste type, Powder-liquid mix type, Eendodontic sealer, Intraosseous implantation 서론 MTA 는우수한밀폐능력, 장기적예후, 치수반응및조직 재생을자극하는능력등을가지며경화후세포독성이감소 * Correspondence: 임범순 (ORCID ID: 0000-0003-3112-0227) 서울시종로구연건동 28 서울대학교치과대학치과생체재료과학교실 Tel: +82-2-740-8692, Fax: +82-2-740-8694 E-mail: nowick@snu.ac.lr Received: Jul. 26. 2017; Revised: Jul. Sep. 06. 2017; Accepted: Sep. 06. 2017 하는경향이있어생체적합성도우수하여근관치료분야인천공수복, 치근단충전, 치수복조, 근첨형성술, 유구치치수절단술및근관충전등에적용되고있다 (Torabinejad 등, 1993; Paranjpe 등, 2010). 또한 MTA는생체활성특성으로치근단조직치유를촉진할수있고, 수산화인회석결정생성으로경조직재생도가능할수있으며 (Gandolfi 등, 2010; Salles 등, 2012), 높은 ph에의한항균효과도기대할수있다고한다 (Hsieh 등, 2009). 229
MTA의조작성문제를개선하기위한다양한연구들이진행되고있다. 멸균수로혼합한 ProRoot MTA의경화시간은 70분정도였는데, Na 2 HPO 4, CaCl 2, CaN/N 또는 CaF 등을첨가한용액으로혼합하면경화시간을 7~35 분으로단축시킬수있다는연구결과들이보고되었으며 (AlAnezi 등, 2011; Ranjkesh 등, 2016), 이경우에도 MTA의우수한생체적합성은유지할수있다고한다 (AlAnezi 등, 2011). 또한, calcium lactate gluconate, methyl cellulose, hydroxypropyl cellulose 및 polyvinyl alcohol 등을첨가한수용액으로 MTA를혼합하여조작성등을개선시키는방법이제시되었고 (Anand 등, 2014; Noh 등, 2015; Baba와 Tsujimoto, 2016), glycerol, polyethylene glycol 또는 propylene glycol 등을첨가하여 MTA의조작성을개선하려는연구들도보고되었다 (Holland 등, 2007; Persson 과 Engqvist, 2011; Duarte 등, 2012; Salem Milani 등, 2013; Natu 등, 2015; Guimarães 등, 2015). 점성의알코올화합물질인 propylene glycol 은발암성및유전독성이없는미국 FDA에서승인받은안전한식품첨가물이며 phenytoin 또는 diazepam 등과유사한용도로약품의첨가제로사용되고있다 (Catanzaro 와 Smith, 1991). 또한 propylene glycol 은일반근관내존재하는병원균에대한항균성이있고, 증류수보다상아질침투력이우수하여 (Bhat 와 Walkevar, 1975; Cruz 등, 2002) 수산화칼슘 [Ca(OH) 2 ] 제재또는근관충전용실러의조작성과항균성을증진하기위한매개물 (vehicle) 로적용되고있다 (Felippe 등, 2005; Nunes와 Rocha, 2005; Chang 등, 2010; Ximenes 와 Cardoso, 2012). 최근에는증류수에 propylene glycol 을첨가하여혼합한 MTA를대상으로흐름성, ph, 경화시간, 수산화칼슘유출및압축강도등을평가한연구결과들이보고되었는데, 대부분증류수에 20 % propylene glycol 을첨가한용액으로혼합한 MTA에서가장적절한물성이관찰되었다고하였다. 특히, propylene glycol을추가함에따라 MTA의흐름성과경화시간은증가되었고, ph와칼슘이온유출도증가하는양상을보였으며, 압축강도와표면경도는감소되었다고하였지만생체적합성에는영향을주지않은것으로보고되었다 (Duarte 등, 2012; Salem Milani 등, 2013; Ghasemi 등, 2016; Marciano 등, 2016b). 또한 MTA에광중합형레진을추가하여개발한변형-MTA 제품 (TheraCal; Bisco, Schamburg, IL, USA) 도소개되었다 (Gandolfi 등, 2012; Karadas 등, 2016). MTA에대한 in vivo 조직반응을평가하기위하여대부분실험동물을이용한피하이식시험을실시하였다. MTA와접촉한조직주변에서수산화인회석 (Reyes-Carmona 등, 2009) 또는탄산인회석 (Holland 등, 2002b) 결정상형성이유도되는것이관찰되었는데, MTA에의한석회화조직구조를관찰하기위하여많은연구들에서 von Kossa기법이사용되었다 (Holland 등, 2001; Holland 등, 2002a). 피하이식한다음 7일이경과한시점에서 MTA 주변으로 von Kossa positive 구조들이생성되는것이관찰되었으며, 장기간이식된실험군에서는더큰석회화구조들이관찰되었다고하였다 (Holland 등, 1999; Holland 등, 2002a). Gray-MTA (GMTA) 와 white-mta (WMTA) 를이식한주위에서대부분석회화구조들이관찰되었지만 (Moretton 등, 2000; Holland 등, 2001; Holland 등, 2002b; Yaltirik 등, 2004), 석회화구조형성이관찰되지않은연구들도보고되었다 (Shahi 등, 2006; Sumer 등, 2006; Vosoughhosseini 등, 2008; Prescott 등, 2008). 골이식시험을실시한연구들에서 MTA에대한골반응이피하이식한경우보다상대적으로가볍고적은염증이관찰되었다고한다. Sousa 등 (2004) 은 MTA, 산화아연유지놀 (ZOE) 및광중합형컴포짓트레진등에대한 guinea pig의골반응을비교하였는데, 이식한다음 4주가경과한시점에서 MTA 주변에서는반응이없거나미약한것으로평가된반면, ZOE 주변은괴사, 골흡수, 단핵의염증세포와이물거대세포의침윤을보였으며, 광중합형컴포짓트레진이식부위에서는중등도의만성염증침윤이관찰되었다고하였다. 그러나이식후 12 주가경과한시점에서골내이식부에서의조직반응을관찰한결과실험군들모두적절한생체적합성을보였다고하였다. 임상에서는 ProRoot MTA (Dentsply, Johnson City, TN, USA) MTA-angelus (Angelus, Londrina, PR, Brazil), Bioaggregate (Innovative Bioceramix, Vancouver, Canada), Micromega MTA (Micromega, Besancon Cedex, France) 및 Biodentine (Septodont, Saint-Maurdes-Fosses, France) 등의수입제품들과 MTA 특허가 2013 년만료된이후국내독자기술로개발된 Ortho MTA와 Retro MTA (BioMTA, Seoul, Korea), Endocem MTA와 Endoseal MTA (Maruchi, Wonju, Gangwon-do, Korea) 및 Well-Root ST (Vericom, 230
Chuncheon, Gangwon-do, Korea) 등의국산제품들이사용되고있다. 대부분의 MTA 제품들은분말과용액을혼합하여사용하는방식으로혼합조건과방법에따라최종 MTA 물성에차이가있을수있다. 그러나최근에소개된 Endoseal MTA 또는 Well-Root ST 등과같은연고 (paste) 형제품들은제조사에서혼합하여제조한 MTA를시린지에충전한제품으로처음사용하는초보자의경우에도사용이용이하고항상균일한물성을기대할수있는장점이있다 (Kwon 등, 2017). 이러한연고형 MTA 제품의구성성분에대한상세한정보는제조사에서제시하지않고있지만, hydroxypropyl cellulose 또는 propylene glycol 등이첨가된것으로추정할수있다. 그러나분말- 용액혼합형 MTA와달리이러한연고형 MTA의물성과생물학적안전성을평가한연구들은아직많지않은실정이다. 이전의연구에서는분말- 용액혼합형 MTA와연고형 MTA의물성을비교하여조작성향상에따른다른물성의변화가있는지비교평가하였고 (Kwon 등, 2017), 본연구에서는물성실험과, 분말- 용액혼합형 MTA, 연고형 MTA 및레진계근관충전용실러를토끼의경골에이식하여생물학적특성을비교평가하고자하였다. 재료및방법 1. 연구재료연구재료로는연고형 MTA인 Well-Root ST와분말-용액 혼합형 MTA인 ProRoot MTA를사용하였고, 현재임상에서많이사용되고있는레진계근관충전용실러인 AH Plus를대조군으로사용하였다 (Table 1). 동물실험에는생후 9~20개월되고무게 3.0~3.5 kg 범위인토끼 (New Zealand white male rabbit) 8 마리를실험에사용하였는데, 실험동물은실온에서고형사료와물을이용하여 1주이상실험동물실에서사육하며관찰한다음이식시험에사용하였다 (ISO 10993-2: 2006). 2. 연구방법 1) 시료이식이식시험은 ISO 10993-6:2016 (Tests for local effects after implantation, Annex C and E. Test method for implantation in bone, and Examples of evaluation of local biological effects after implantation) 과식품의약품안전처고시제 2013-64 호 ( 의료기기생물학적안전에관한공통기준규격 : 제 6장이식시험. 부록 D와 E, 골조직내이식시험방법과이식후나타나는국소적인생물학적인영향평가의예 ) 에따라실시하였다. 토끼를 Ketamin 0.05% 와 Rompun 2% 로전신마취한후경골 (tibia) 의피질부분을노출시킨다음 surgical kit를사용하여양쪽에각각 3개또는 2개씩시료를식립하기위한구멍 ( 지름 2 mm 깊이 6 mm) 을형성하였다. Well-Root ST와 ProRoot MTA를각각 4마리의토끼에 10개씩이식하였으며, 실험군을이식한반대편에대조군으로 AH Plus를 10개씩이 Table 1. Materials used in this study Material Composition Lot No. Manufacturer Well-Root ST calcium silicate compound, zirconium oxide, titanium oxide, calcium sulfate dihydrate, calcium sodium phosphosilicate, thickening agents WR590100 Vericom, Chuncheon, Gangwon-do, Korea ProRoot MTA tricalcium silicate, dicalcium silicate, tricalcium aluminate, tetracalcium aluminoferrite, bismuth oxide, calcium sulfate 0000092761, 0000100365 Dentsply, Johnson City, TN, USA AH Plus (control) epoxy resin, adamantane amine, N,N-dibenzyl-5-oxanonane-diamine-1,9, TCD-diamine, calcium tungstate, zirconium oxide, aerosil, iron oxide 1507000612 Dentsply, Konstanz, Germany 231
식하였다. 제조사의사용방법에따라각시료를이식할구멍에충전하여식립하였다. 2) 임상적평가시료를이식한후토끼의상태를최소주 1 회씩육안으로관찰하여염증소견, 합병증및특이사항유 무등을확인하였다 (Table 2). 이식부위및주변의혈종 (haematoma), 부종 (oedema), 캡슐화 (encapsulation) 등을기록하였다. 3) 조직학적평가시료를이식하고 12주가경과한시점에서토끼에 KCl (2 mmol/kg) 을정맥주사하여안락사시킨다음이식부위를육 안으로관찰하였다. 또한, 이식한조직을채취하여각각 10% formaldehyde solution 에 48시간고정하였고, formic acid로탈회한다음파라핀포매하여경화시켰다. 이때형성된골이식부위보다넓은영역을 serial cross-section 하여조직절편을제작하고, Hematoxylin & Eosin 염색을시행한후광학현미경으로조직학적평가를실시하여생물학적반응변수에따라결과를평가하였다. 염증의정도와염증세포의수및분포형태 [ 다형핵백혈구 (polymorphonuclear cell), 림프구 (lymphocyte), 플라즈마세포 (plasma cell), 대식세포 (macrophage), 다중핵세포 (multinucleated cell)], 세포핵의잔해또는모세혈관벽의파괴로인한괴사의발생, 그리고신생혈관화, 섬유화, 지방세포의침투등과같은조직변화 Table 2. Evaluation of clinical observation Index Clinical Sign Index Clinical Sign - No abnormalities / Survival G Prostration A Death H Hair loss B Abnormal gait I Tremor C Piloerection J Salivation D Muddy stool K Clonic convulsion E Nasal bleeding L Tonic convulsion F Diarrhea M Decreased spontaneous locomotion Table 3. Histological evaluation system (cell type/response) Cell type / response Polymorphonuclear cell 0 Score 0 1 2 3 4 Rare, 1-5/phfa 5-10/phf Heavy infiltrate Lymphocytes Plasma cells Macrophages Giant cell Rare, 1-2/phf Packed 3-5/phf Sheets Necrosis Minimal Mild Moderate Severe a phf = per high-powered (400 ) field. 232
Table 4. Histological evaluation system (tissue response) Response Score 0 1 2 3 4 Neovascularization 0 Minimal capillary proliferation, focal, 1 to 3 buds Groups of 4 to 7 capillaries with supporting fibroblastic structures Broad band of capillaries with supporting fibroblastic structures Extensive band of capillaries with supporting fibroblastic structures Fibrosis 0 Narrow band Moderately thick band Thick band Extensive band Fatty infiltrate 0 Minimal amount of fat associated with fibrosis Several layers of fat and fibrosis Elongated and broad accumulation of fat cells about the implant site Extensive fat completely surrounding the implant 등을정량적으로평가하여기록하였다 (Table 3과 Table 4). 결과실험군과대조군을실험동물에이식후주기적으로실험동물의임상증상을관찰한결과, 특이할만한합병증이나염증소견등어떠한임상적이상증상도관찰되지않았다. 실험군과대조군의이식기간종료후이식부위는육안평가및조직학적평가를통하여관찰하였다. 육안평가기준에따라관찰한결과대조군과실험군의이식부위주변조직에서혈종 (hematoma), 부종 (edema), 캡슐화 (encapsulation) 등은관찰되지않았다. Well-Root ST를이식한군에서신생골이가장많고균일하게형성된것을관찰할수있었다. 두번째로신생골이잘형성된군은 ProRoot MTA를이식한군이었으며, 두가지실험군은모두이식재의주변으로미세혈관침투와골형성이일어나는것을관찰할수있었다 (Fig. 1). AH Plus 를이식한대조군의경우림프구및다형핵백혈구의심한침윤이관찰되었지만 (Fig. 2a), Well-Root ST를이식한군에서는결손부와평행하게형성된신생골을확인할수있었는데, AH Plus를이식한대조군과비교하여증가된골형성을볼수있었으며광화도증가와신생골및신생혈관의성숙을관찰할수있었다 (Fig. 2b). ProRoot MTA를이식한군도연속성 을보이는신생골형성이관찰되었으나 Well-Root ST를이식한군에비해서는형성된신생골의양이적은양상을볼수있었으며, 증등도의섬유화도관찰되었다 (Fig. 2c). 본연구에서분석한대조군과실험군의전체시편 ( 각각 10개씩 ) 사진은 Fig. 3에정리하였다. 조직병리학자의판독소견에따른조직학적평가결과실험군의 irritation ranking score 값은 0 으로대조군과비교하여비자극성 (non-irritant) 으로평가되었다 (Table 5과 Table 6). 따라서시험군의시료는잠재적인생물학적위해반응이없다고사료되므로, 이식시험기준에적합하다고판정되었다. 고찰 1930 년대에소개된수산화칼슘제재 [Ca(OH) 2 ] 는치과임상에서치수복조, 치수절단, 치근첨형성유도, 근관내소독, 지속적인삼출물조절및치아재이식후의치근흡수치료등다양하게이용되고있다. 수산화칼슘제재의작용기전은명확하게밝혀지지않았으나칼슘과수산화이온이관련된것으로보인다. 수산화칼슘제재의주요성분은칼슘이온으로세포자극, 이주, 증식및분화를유도하고석회화의개시재로작용하여상아질교를형성하여치수를보호하는역할을한다 (Farhad 와 Esfahan, 2005). 수산화이온이상아세관을통 233
하여확산되어치근주위 ph를증가시켜알칼리성환경을제공하고, 높아진 ph는세균을죽이고파골세포의활성을억제한다고한다 (Massarstrom 등, 1986). 석회화과정에서 calcium carbonate granulation 이 von Kossa-positive calcium salt granulation 의침착을유도하여조직의치유를촉진하고상아모세포의분화와상아질침착에기여할수있다고하는데, calcium carbonate granulation 은 Ca 2+, CO 2 및 CO 3- 이결합하여형성된다고한다 (Holland 등, (a) (b) (c) Figure 1. Histological analysis (left = 12.5 magnification, right = 100) of experimental groups after 12 weeks: a) AH Plus, b) Well- Root ST, c) ProRoot MTA (NB = new bone, PB = pre-existing bone, BM = bone marrow, = implanted material). 234
(a) (b) (c) Figure 2. Histological analysis ( 200 magnification) of experimental groups after 12 weeks: a) AH Plus, b) Well-Root ST, c) ProRoot MTA. 1982). Holland 등 (1982) 은이때사용되는칼슘이온이주변조직에서용출된것이아니라사용한수산화칼슘제재에서유리되는것이라고하였는데, 이러한칼슘이온은 MTA에서도유리될수있다. 수산화칼슘은 MTA의구성성분이아니지만, MTA 성분인 calcium silicate 가수화반응하면 calcium silicate hydrate와수산화칼슘상이생성되므로수산화칼슘제재와유사한기전으로경조직형성에기여할수있다고한다 (Holland 등, 1999; Faraco 와 Holland, 2001). 또한이때높은알카리성을보이는데 (Camilleri 등, 2008), 생성된높은알칼리성이 MTA의항균성에중요한역할을한다 (Parirokh 와 Torabinejad, 2010). 많은연구들에서 MTA를직접치수복조제로적용하여좋은효과가관찰되었다고하였고, 수산화칼슘제재와비교하여염증이적고상아질교형성도우수하였다고하였다 (Ford 등, 1996; Faraco 와 Holland, 2001; Hwang 등, 2009). 생체조직과유사한수분함유하고균일한전도성매질역할을할수있는 hydrogel 은분말또는입자들의유지와이동을위한적절한기질로사용할수있으며, 골대체재또는골재생을위한 scaffold 등에도적용할수있다 (Giannoni 등, 2016). 조직공학용 hydrogel 로적용될수있는천연폴리머로는 collagen, hyaluronan, alginate, chitosan 및 fibrin 등이있고, 합성폴리머로는 polyethylene glycol, polyvinyl alcohol, polypropylene fumarate, cellulose derivatives ( 예, hydroxypropyl methyl cellulose, Pluronic F-127 및 polypeptides 등이있다 (Lee와 Mooney, 2001). 점성폴리머인 carboxymethylcellulose (CMC) 와 hydroxypropylmethylcellulose (HPMC) 는약리물질과생체재료의운반과유지등을위하여사용되고있다. 다양한임상연구에서 CMC와 HPMC는 bone morphogenetic protein (BMP-2), osteogenic protein-1 (OP-1/BMP-7) 등과같은골전도성물질의운반매개물로사용할수있을뿐아니라 calcium sulfate 또는소뼈유래수산화인회석등과같은골대체재의이송수단으로도사용할수있다고알려져있으며 (Agis 등, 2010), 오래전부터치과용수산화칼슘제제에적용된바있다 (Fava 와 Saunders, 1999). Baba 와 Tsujimoto (2016) 는점도가낮은 methylcellulose (MC) 와 hydroxypropylcellulose (HPC) 2 wt% 를 MTA에첨가하여 ISO 6876:2012 에따라평가한결과 235
Table 5. Histological evaluation of Well-Root ST Response Groups Control (AH Plus) Well-Root ST Sample # Inflammation 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 PMN cell 0 2 3 3 3 3 2 3 3 3 0 0 0 0 0 0 0 0 0 0 Lymphocytes 2 2 3 3 3 3 2 3 3 3 0 1 0 0 0 1 0 0 0 0 Plasma cells 2 2 3 2 2 0 3 0 1 0 0 0 1 0 0 0 0 0 2 0 Macrophages 2 2 2 3 2 0 2 0 0 0 0 0 0 0 1 0 0 1 0 1 Giant cell 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Necrosis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SUB TOTAL ( 2) 12 16 22 22 20 12 18 12 14 12 0 2 2 0 2 2 0 2 4 2 Neovascularisation 0 1 1 2 2 1 1 1 1 1 1 2 1 2 3 2 1 0 2 3 Fibrosis 1 0 0 0 1 1 0 1 0 0 0 0 1 0 0 0 0 0 0 0 Fatty infiltrate 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SUB TOTAL 1 1 1 2 3 2 1 2 1 1 1 2 2 2 3 2 1 0 2 3 Total 13 17 23 24 23 14 19 14 15 13 1 4 3 2 5 4 1 2 6 5 Group Total 175 33 Averagea Well-Root ST ( 3.3 ) - Control ( 17.5 ) = -14.2 ( 0 ) Traumatic necrosis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Foreign debris 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 No. sites examined 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Well-Root ST non-irritant (0.0 up to 2.9) slight irritant (3.0 up to 8.9) moderate irritant (9.0 up to 15.0) severe irritant (>15) a Used to determine reactivity ranking shown below as the conclusion. A negative difference is recorded as zero. 흐름성, 경화시간및칼슘이온용출등은증가하였다고하였다. Ber 등 (2007) 은 methylcellulose 만 1~3 wt% 첨가할경우경화시간은약간증가하는양상을보인반면, methylcellulose 와 CaCl 2 를함께첨가할경우경화시간을 50~70% 정도감소시킬수있다고하였다. Noh 등 (2015) 은 PVA를 1% 이하로 첨가할경우조작성이향상되지않았으며, 7% 이상첨가할경우에는균일하게혼합하기어려우므로 3~5% 정도만추가하면 MTA의물성에영향을주지않고조작성을개선할수있다고하였다. Polyethylene glycol (PEG) 은 ethylene oxide의중합체로 236
Table 6. Histological evaluation of ProRoot MTA Groups Control (AH Plus) ProRoot MTA Sample # Response Inflammation 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 PMN cell 0 2 2 2 2 2 2 2 2 3 1 0 1 0 2 1 1 1 1 1 Lymphocytes 2 1 3 2 3 2 2 3 2 3 1 1 1 2 1 1 0 1 0 0 Plasma cells 1 2 2 2 2 2 2 1 1 0 1 1 1 0 0 1 0 1 0 0 Macrophages 2 2 2 2 2 0 2 1 0 0 0 1 1 2 1 1 0 2 2 1 Giant cell 0 0 0 1 1 0 1 0 0 0 1 2 1 0 1 1 1 0 1 1 Necrosis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 SUB TOTAL ( 2) 10 14 18 18 20 12 18 14 10 12 8 10 10 8 10 10 4 10 8 6 Neovascularisation 0 1 1 2 2 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 Fibrosis 1 0 0 0 1 1 0 1 0 0 0 1 0 1 0 0 0 1 0 0 Fatty infiltrate 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 0 SUB TOTAL 1 1 1 2 3 2 1 2 1 1 1 3 2 2 1 1 1 2 1 1 Total 11 15 19 24 23 14 19 16 11 13 9 13 12 10 11 11 5 12 9 7 Group Total 165 99 Averagea ProRoot MTA ( 9.9 ) - Control ( 16.5 ) = -6.6 ( 0 ) Traumatic necrosis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Foreign debris 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 No. sites examined 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 Well-Root ST non-irritant (0.0 up to 2.9) slight irritant (3.0 up to 8.9) moderate irritant (9.0 up to 15.0) severe irritant (>15) a Used to determine reactivity ranking shown below as the conclusion. A negative difference is recorded as zero. 물, 메탄올, 벤젠, 디클로로메탄등에용해될수있고, 물과친화력이있는분자구조물질이며, 비이온화계면활성제로만들면독성이없고완전불활성상태여서 (Shethe 와 Leckband, 1997), 저분자량 PEG는현재성인또는소아의 장-세척용으로현재사용하고있다. 또한 PEG은연고형수산화칼슘제재의매개물로적용된바있다 (Fava와 Saunders, 1999). Propylene glycol (PG) 은무색 투명하고냄새가없거나미세한냄새가있을수있으며약간쓴맛과단맛이있는 237
흡습성 액체로 점성이 있어 외관은 글리세린과 유사하다. 물 큰 차이가 없으며, 매우 많은 양 (LD50 값이 15 g/ 이상)에 또는 알코올과 잘 혼합되고 클로로포름, 아세톤 등의 유기용 서만 독성이 발생된다고 한다(Center for the Evaluation of 매와도 혼합되는 특성을 가지고 있으며 독성도 아주 약해 식 Risks to Human Reproduction, 2004). PEG와 함께 PG도 오 품첨가물로 많이 사용되는 있다(Mohamed와 Stainsby, 래전부터 연고형 수산화칼슘제재 등의 매개물로 사용되었다 1984). PG의 통상적인 하루 평균 섭취량은 2,400 mg 수준인 (Nalawade 등, 2016). MTA의 물성을 개선하기 위하여 PG를 것으로 보고되었는데, 인간과 동물에 대한 일반적인 독성은 추가한 MTA의 특성을 평가한 연구들이 보고되었는데, PG를 # CTL (AH Plus) EXP-1 (Well-Root ST) EXP-2 (ProRoot MTA) 1 2 3 4 5 Figure 3. Overall histological images 12 weeks: CTL (AH Plus), EXP-1 (Well-Root ST), EXP-2 (ProRoot MTA) at 12.5 magnification. 238
추가한 경우 MTA의 점조도 (consistency), 조작성 및 push- 영향을 주지 않았다고 하였다(Holland 등, 2007). Duarte 등 out 결합력 등이 개선되었고, 경화시간이 단축되었으며, 경화 (2012)은 증류수에 PG (0~100 wt%)를 첨가한 용액으로 초기 단계에서 ph와 칼슘이온 용출을 증가시켰다는 결과가 MTA-Angelus 분말을 혼합하여 물리 화학적 특성을 평가한 결 발표되었다(Hsieh 등, 2009; Brito-Júnior 등, 2010; AlAnezi 과 증류수에 PG를 첨가하면 ph와 칼슘이온 용출에는 영향을 등, 2011; Duarte 등, 2012; Salem Milani 등, 2013). 또한 MTA 주지 않았지만, PG 첨가량이 증가할수록 혼합물의 경화시간 에 PG를 추가하여도 MTA의 우수한 생체적합성은 유의한 차 이 지연되기 때문에 필요할 경우에는 증류수에 PG를 20 wt% 이를 보이지 않았으며, 세포에 대한 생물학적 반응에도 나쁜 첨가할 것을 추천하고 있다(Marciano 등, 2016a; Marciano # CTL (AH Plus) EXP-1 (Well-Root ST) EXP-2 (ProRoot MTA) 6 7 8 9 10 Figure 4. Overall histological images 12 weeks: CTL (AH Plus), EXP-1 (Well-Root ST), EXP-2 (ProRoot MTA) at 12.5 magnification. 239
등, 2016b). Salem Milani 등 (2015) 은 PG를첨가한용액으로혼합한 MTA의표면경도가감소되는양상을보였다고하였고, Ghasemi 등 (2016) 은증류수에 PG를 20 wt% 첨가한용액으로혼합한 MTA의압축강도가크게감소되는문제를제기하였다. Marques 등 (2013) 은 PG를추가한 Portland cement, MTA Fillapex, iodoform 을추가한산화아연유지놀 paste를쥐에피하이식하여조직반응을평가한결과 PG를추가한 Portland cement 에서우수한생체적합성이관찰되었다고하였다. Natu 등 (2015) 은증류수에 PG를 50% 또는 20% 비율로추가한 MTA의물성을평가한결과흐름성외에는특별하게개선된특성이없었으며, MTA의조작성개선을위해서는증류수에 20% PG 추가를고려해볼만하다고하였다. Poggio 등 (2015) 은광중합형레진을추가하여개발한변형-MTA 제품인 TheraCal 의세포독성을 Dycal, Calcicur, Calcimol LC, ProRoot MTA, MTA-Angelus 및 Biodentine 등의근관충전제와비교하여평가한연구에서 TheraCal 은수산화칼슘제재와유사하게관찰되어 MTA 성분으로구성된장점을보이지못하였다고하였다. Holland 등 (2001, 2002a) 은 PC (Portland Cement), CH (Calcium hydroxide), MTA를피하이식하여비교한연구에서각재료들은유사한피하반응을보였으며, 생체조직내에이산화탄소가있는경우각재료에서유리된칼슘이온들의반응으로유사한석회화조직이생성된다고하였다. Moretton 등 (2000) 은 GMTA를쥐에피하이식한경우 EBA를이식한경우보다유의하게많은염증이유발된것이관찰되었는데, 초기에는응고괴사, 이영양성석회화와함께심한반응을보였고시간이경과함에따라이상반응은진정되었으나피하이식된 MTA 주변에서골형성은관찰되지않아 MTA가피하조직에서는골유도성 (osteoinductive) 이없다고하였다. GMTA 와 WMTA 에대한피하반응을비교한연구에서도이식 3일후에는 WMTA 가 GMTA 보다유의하게적은염증을일으켰고 7일후에는 WMTA 가 GMTA 보다더많은염증이관찰되었다는연구보고가있으며 (Shahi 등, 2006), 다른연구에서는 GMTA와 WMTA 에대한염증반응에서유의한차이가없었다는상반된결과가보고되었는데이러한차이는조직병리학적평가법이상이하기때문에발생한것이라고하였다 (Vosoughhosseini 등, 2008). 쥐의피하조직에 PC와 WMTA 를이식한경우모두경미하거나중간정도의조직반응을보였고, 이식후 7일과 14일에더많은호산구를보인 CPM (modified Portland Cement) 에비하여 MTA 주변에는더많은조직괴사와거대세포형성을보였다고하였고 (Martínez Lalis 등, 2009), 이식된모든 MTA 시료주변에서석회화침전물이관찰되었다 (Gomes-Filho 등, 2009). 이연구들은 MTA 에대한피하반응이이식초기에는중등도의반응을보이지만시간이경과되면서진정되는양상을보였다고하였다. 골내반응에대한연구들은 MTA에대한골반응이피하이식한경우보다상대적으로가볍고염증이적다는것을보였다. Sousa 등 (2004) 은 ZOE, 광중합형컴포짓트, MTA에대한 guinea pig의골반응을비교한연구에서이식 4주후 MTA 의반응이없거나미약한것으로평가된반면 ZOE는괴사, 골흡수, 단핵의염증세포와이물거대세포의침윤이관찰되었다고하였으며, 광중합형컴포짓트시료는이식된재료근처에중등도의만성염증침윤을보였다고하였고, 골내이식시험에서재료에대한조직반응은 12주후진정되어모든실험재료들은생체적합특성을보였다고하였다. Torabinejad 등 (1995b) 은 guinea pig의하악골에이식된 SuperEBA 와 MTA 에대한조직반응을평가한연구에서 MTA 에대한골반응이 SueprEBA 에대한경우보다조금약하였다고보고하였다. 다른연구에서 Torabinejad 등 (1998) 은아말감, SuperEBA, IRM, MTA에대한 guinea pig 경골의반응을비교한결과 MTA에서가장긍정적인반응이관찰되었다고보고하였다. Moretton 등 (2000) 은 MTA와 EBA의피하이식반응과골내이식반응을비교하였는데, 초기에는골형성패턴이유사했지만, 60일경과한경우에는 MTA보다 EBA 주변에서더큰골형성이관찰되었고, 두재료모두골전도성을가진다고보고하였다. MTA를쥐의골조직속에매식했을때주위에골조직이침착되었고, 쥐의연조직에매식했을때는주위에염증조직없는섬유조직으로캡슐화된것으로볼때 MTA 자체가 BMP-2 처럼골을생성할수있는능력은없지만골형성을돕는 osteoconductive 역할은할수있는것으로예상된다고하였다 (Moretton 등, 2000). Cintra 등 (2006) 은쥐의치조골에대한 MTA와새로운 calcium hydroxide containing sealer (MBPc) 의반응을비교분석하였는데두재료간에는유의한차이가없었다고하였다. 240
MTA를치근단역충전재또는치근천공수복을위하여개 (beagle) 에게적용한실험에서 MTA 주변으로는염증세포가거의관찰되지않았으며, 골조직의회복과치주인대막및백악질의생성이관찰되었다고하였다 (Baek 등, 2005; Asgary 등, 2008). MTA가세포의증식과분화를증진시키기위하여특정한 cytokine 의분비를증가시킨다는결과도보고되었는데, cytokine 은염증반응의매개체들로 MTA가세포배양시존재하면세포들이이런매개체를더많이분비하는것으로추정할수있다고한다 (Koh 등, 1997). 일부연구에서는 MTA 와함께세포배양한경우에서 MTA 없이세포배양한대조군보다일정시간내에더많은세포가증식되었다는결과도보고되어 MTA가세포증식을활성화시킬수있는가능성을제시하기도하였다 (Huang 등, 2003). 골모세포를 MTA와함께배양해서골을형성하는 marker (alkaline phosphatase, osteopontin, osteonectin 등 ) 가더많이발현되는가를관찰한연구에서 MTA가여러세포들을분화시키는데기여한다는연구결과가보고된바있다 (Perinpanayagam 과 Al-Rabeah, 2009). 골적합성연구에서 Sousa 등 (2006) 은 AH Plus를 guinea pig의턱뼈에이식한다음 4주경과한시점에서평가한결과급성염증반응이관찰되었다고하였고, 12주경과한시점에는중등도의만성염증특성이관찰되었다고하였다. Assmann 등 (2015) 은 MTA를함유한 MTA Fillapex (Angelus, Brazil) 와에폭시레진계실러인 AH Plus (Dentsply, Germany) 를쥐의대퇴골에 7일, 30일및 90일간식립하여골조직과의반응을평가하였는데, AH Plus와비교하여 MTA를함유한 MTA Fillapex 가초기골조직복원에더효과적이지않았지만, 시간이경과됨에따라염증반응이감소되고원래골조직구조가복원되는것이관찰되어두가지재료모두유사한생체적합성이있는것으로보였다고하였다. 이식된실러 (sealer) 주변에서는대조군에서관찰되지않은섬유질응집이관찰되어골재생이가능한유기반응은실러주변에국한된것으로추론할수있다. 대조군에서는경조직장벽형성이 30일내에완벽하게진행된반면, 실러를이식한경우에는이물질이소멸되는기간이필요하여 90일경과한후에야관찰되었다. 본연구에서 ISO 10993-6:2016 에서제시한시험법에따라연고형 MTA, 혼합형 MTA 및에폭시레진계실러의골내이 식시험을실시하여비교평가한결과연고형 MTA도기존에많은연구결과가보고된에폭시레진계실러보다는우수하고, 분말- 용액혼합형 MTA와는유사한특성이관찰되어임상적용에문제가없을것으로보였다. 결론본논문에서는 ISO 10993-6:2016 에따라실험군으로연고형 MTA와분말- 용액혼합형 MTA, 대조군으로레진계근관충전용실러인 AH Plus 를토끼의경골에 12주간이식하여골내이식시험을실시하여비교한결과최근에소개된연고형 MTA는기존분말-용액혼합형 MTA의대체용으로사용하기충분한생물학적특성을가지는것으로보였다. 참고문헌 Agis H, Beirer B, Watzek G, Gruber R (2010). Effects of carboxymethylcellulose and hydroxypropyl methylcellulose on the differentiation and activity of osteoclasts and osteoblasts. J Biomed Mater Res A 95:504-509. AlAnezi AZ, Zhu Q, Wang YH, Safavi KE, Jiang J (2011). Effect of selected accelerants on setting time and biocompatibility of mineral trioxide aggregate (MTA). Oral Surg Oral Med Oral Pathol Oral Radiol Endod 111:122-127. Anand S, Taneja S, Kumari M (2014). Effect of accelerants on the immediate and the delayed sealing ability of mineral trioxide aggregate when used as an apical plug: An in vitro study. J Conserv Dent 17:45-48. Asgary S, Mohammad JE, Masoud P, Farzin G, Hessam R (2008). A comparative study of histologic response to different pulp capping materials and a novel endodontic cement. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 108:609-614. Assmann E, Böttcher DE, Hoppe CB, Grecca FS, Kopper 241
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연고형 Mineral Trioxide Aggregates (MTA) 와분말 - 용액혼합형 MTA 의골내이식시험비교연구 권영대, 석수황, 이상혁, 임범순 * 서울대학교치의학대학원치과생체재료과학교실 본연구에서는분말-용액혼합형 MTA와연고형 MTA의물성을비교하고, 분말-용액혼합형 MTA, 연고형 MTA 및레진계근관충전용실러를토끼의경골 (tibia) 에 12 주간이식하여그결과를비교평가하고자하였다. ISO 10993-6:2016 에따라골내이식시험을실시하였는데, 전신마취시킨토끼의경골피질부분을노출시킨다음양쪽에각각 3개또는 2개씩시료를식립하기위한구멍 ( 지름 2 mm 깊이 6 mm) 을형성한후제조사의사용방법에따라각시료를이식할구멍에충전하였다. 연고형 MTA인 Well-Root ST와분말-용액혼합형 MTA인 ProRoot MTA를실험군으로하였고, 임상에서많이사용되고있는레진계근관충전용실러인 AH Plus를대조군으로이식하였다. 시료를이식한후에는토끼의상태를최소주 1회씩육안으로관찰하여염증소견, 합병증및특이사항유 무등을확인하여기록하였고, 시료를이식하고 12 주가경과한시점에서토끼를안락사시킨다음이식부위를육안평가와조직학적평가를실시하였다. 연고형 MTA를이식한실험군에서신생골이가장많고균일하게형성된것이관찰되었고, 분말-용액혼합형 MTA를이식한실험군에서어느정도신생골이형성된것이관찰되어 MTA 주변으로미세혈관침투와골형성이진행됨이관찰되었다. 에폭시레진계근관충전용실러를이식한대조군의경우림프구및호중구의심한침윤이관찰되었다. 조직학적평가결과연고형 MTA와분말-용액혼합형 MTA는대조군인 AH Plus와비교하여잠재적인생물학적위해반응이없는 비자극성 (non-irritant) 으로평가되었다. 최근에소개된연고형 MTA는기존분말-용액혼합형 MTA의대체용으로사용하기충분한생물학적특성을가지는것으로보였다. 색인단어 : MTA, 연고형, 분말 - 용액혼합형, 근관충전용실러, 이식시험 246