본연구에서는탈락후다양한보관매체내에서일정기간동안보관용액에보관한유치의치수에서세포를배양하여, 유치치수의세포가보관용액의종류와기간에영향을받는지에대하여연구하였다. Ⅱ. 연구재료및방법 1. 유치의획득및보관이번연구는 2012년 1월부터 2012년 12월까지연세대학교치과대학기관윤리위원

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1 J Korean Acad Pediatr Dent 41(1) 2014 ISSN (print) ISSN (online) Effect of Storage Media and Duration on Pulpal Cell Viability in Exfoliated Deciduous Teeth Jiwon Park 1, Jeseon Song 1,2, Jaeho Lee 1,2, Seongoh Kim 1,2, Mijeong Jeon 2, Hansung Jung 3, Heungkyu Son 1,2 1 Department of Pediatric Dentistry, College of Dentistry, Yonsei University, Seoul, Korea 2 Oral Science Research Center, College of Dentistry, Yonsei University, Seoul, Korea 3 Division in Anatomy & Developmental Biology, Department of Oral Biology, College of Dentistry, Yonsei University, Seoul, Korea Abstract If it is possible to preserve and culture cells from exfoliated deciduous teeth in a readily available storage medium within each family, more stem cells would be obtained. This research is about the effect of storage media and time on pulpal cell viability of exfoliated deciduous teeth. 330 exfoliated deciduous teeth were randomly divided into 11 groups; fresh group, dry group, groups stored in cell culture medium (2, 4, 7 days each), in milk (2, 4, 7 days each), and in saline (2, 4, 7 days each). Primary culture of pulpal cells was conducted in each group and the success rates were compared by calculating the number of teeth with viable cells. The result of primary culture shows that the success rate decreases as the time of storage gets longer. There was no statistical difference between groups stored in the cell culture medium, milk, and saline for 2 and 4 days. However, the groups stored in milk and saline for 7 days showed dramatic decrease in success rate compared to the group stored in the cell culture medium. In conclusion, exfoliated or extracted deciduous teeth can be used to culture pulpal cells when they are stored in milk and saline for a certain period of time; however obtaining viable pulpal cells becomes harder as the storage time gets longer. Key words : Storage medium for deciduous teeth, Pulpal tissue, Cell viability, Milk, Saline Ⅰ. 서론흡수탈락된유치의치수조직 1), 발거된선천치의치수 2) 및유치의치주인대 3) 에서줄기세포를얻을수있다고보고되었다. 탈락된유치에서얻은줄기세포는비침습적으로얻을수있으며, 영구치의치수에서얻은줄기세포와비교하였을때더높은증식률, 분화능을보였다는보고 4) 가있었다. 이러한유치의장점때문에최근유치에서얻은조직을간엽성줄기세포의원천으로사용하는연구가활발하게이루어지고있다 1,5-7). 치아를용액에보관하는것에대한연구는영구치의이식이 나재식을위한치아주변인대세포의생존 8-12) 과관련된연구가선행되었으며, 유치의경우에는재식이나이식하는경우가없어현재까지탈락유치의보관용액및조건에따른추출세포의생존률이나특성에대한보고는없는상태이다. 그러나유치줄기세포에대한관심이증가하면서치과및집에서발거된유치추출세포의생존률을높이는방법에대한연구가필요하다고판단되고, 가정에서불시에유치가탈락하였을경우주변에서쉽게구할수있는저장용액에유치를임시로보관한후줄기세포를추출할수있다면지금보다더많은유치를줄기세포연구에이용할수있을것이라고예상된다. Corresponding author : Prof. Heungkyu Son Department of Pediatric Dentistry, Yonsei University College of Dentistry, 250 Seongsanno, Seodaemun-gu, Seoul , Korea Tel: / Fax: / HGSON@yuhs.ac Received July 3, 2013 / Revised November 14, 2013 / Accepted November 14, 2013 This study was supported by a faculty research grant of Yonsei University College of Dentistry for 2013 (No ). These authors equally contributed to this work. 1

본연구에서는탈락후다양한보관매체내에서일정기간동안보관용액에보관한유치의치수에서세포를배양하여, 유치치수의세포가보관용액의종류와기간에영향을받는지에대하여연구하였다. Ⅱ. 연구재료및방법 1.

실험은연세대학교치과대학병원소아치과에내원한환자의진료중에얻게된유치중에서자연탈락이가까운치근 1/3 이하잔존한유전치와유구치, 공간관리교정의목적으로발거한유치를대상으로하였고심한치아우식증으로인한치수감염의가능성이있는치아, 치주염이있는치아, 치근의비정상적인치근흡수나내흡수가존재하는치아, 세포대사에영향을줄수있는특이한전신질환이있는어린이에게서발거한유치는제외시켰다.

L-glutamine (Invitrogen), 10 mm L-ascorbic acid (Sigma, St. Louis, MO, USA), 2.5 μg/ml amphotericin B (Bristol-Myers Squibb, New York, USA) 가첨가된용액을사용하였다.

2 본연구에서는탈락후다양한보관매체내에서일정기간동안보관용액에보관한유치의치수에서세포를배양하여, 유치치수의세포가보관용액의종류와기간에영향을받는지에대하여연구하였다. Ⅱ. 연구재료및방법 1. 유치의획득및보관이번연구는 2012년 1월부터 2012년 12월까지연세대학교치과대학기관윤리위원회로부터승인 ( ) 과환아및보호자의동의를받고서이루어졌으며만 4세에서 14세이하의건강한남자어린이 171명과여자어린이 159명 ( 총 330 명 ) 으로부터한환아당한개의유치를얻어, 총 330개의유치를실험에이용하였다. 실험은연세대학교치과대학병원소아치과에내원한환자의진료중에얻게된유치중에서자연탈락이가까운치근 1/3 이하잔존한유전치와유구치, 공간관리교정의목적으로발거한유치를대상으로하였고심한치아우식증으로인한치수감염의가능성이있는치아, 치주염이있는치아, 치근의비정상적인치근흡수나내흡수가존재하는치아, 세포대사에영향을줄수있는특이한전신질환이있는어린이에게서발거한유치는제외시켰다. 유치를발거한후 11개군에각각 30개씩무작위로배정하였는데, 양성대조군으로발치직후실험한신선군, 음성대조군으로 2일간상온에서건조시킨후실험한건조군, 생리식염수 ( 중외제약, 서울, 대한민국 ), 우유 ( 연세우유, 아산, 대한민국 ), 세포배양액에각각 2일, 4일, 7일동안 4 에서보관한후실험 한실험군 ( 세포배양액, 우유, 생리식염수각각 2, 4, 7일군 ) 으로구분하였다. 세포배양액은 minimum essential medium (- MEM; Invitrogen, Carlsbad, CA, USA) 에 10% fetal bovine serum (FBS; Invitrogen), 200 U/ml penicillin (Invitrogen), 300 μg/ml streptomycin (Invitrogen), 2 mm L-glutamine (Invitrogen), 10 mm L-ascorbic acid (Sigma, St. Louis, MO, USA), 2.5 μg/ml amphotericin B (Bristol-Myers Squibb, New York, USA) 가첨가된용액을사용하였다. 세포배양액, 생리식염수, 우유는 3 ml씩 15 ml conical tube (SPL Life Sciences, Pocheon, Gyeonggi, Korea) 에보관하였으며, 2일마다보관용액을교체해주었다. 2. 유치치수조직의분리및일차배양 (Primary culture) 세포의 1차배양은 outgrowth method를사용하였다. 발거된유치의치수조직은 barbed broach (Mani, Inc, Utsunomiya Toshi-ken, Japan) 를이용하여서치수조직전체를분리해냈다 (Fig. 1A, 1B). 이렇게얻어진치수조직은 1 mm³ 내외의크기로잘게자른후 60 mm culture dishes (BD Falcon, Lincoln Park, NJ, USA) 위에올려놓고 (Fig. 1C) cover glass (Superior, Lauda-Königshofen, Germany) 를덮은후세포배양액을첨가하였다 (Fig. 1D). 상기배양배지를 37 의온도, 5% CO2의습윤환경에서배양하여일주일이지난후관찰하였을때치수조직으로부터자라서나오는세포가관찰되면배양성공으로간주하였다. 관찰시에는광학현미경 (Leica microsystem, Wetzlar, Germany) 을 100배율로사용하였다. A B C D Fig. 1. Primary culture from deciduous teeth. (A) Deciduous tooth from a 10-year-old boy. (B) Pulpal tissue was taken out with barbed broach. (C) Pulpal tissue was cut off to several fragments at size of 1 mm 3 and placed on a 60 mm culture dish (D) Pulpal tissue was covered with cover glass and incubated with culture medium at 37 in a humid atmosphere containing 5% CO 2. 2

유치치수조직의일차배양성공시에는일주일내에전형적인방추형의섬유모세포형태의세포가자라나오는것이관찰되었으며 (Fig. 2A), 배양실패시에는일주일이지난후에도치수조직으로부터자라서나오는세포가관찰되지않았다 (Fig. 2B). 3. 통계분석 (Statistical analysis) 결과분석은 SPSS (19.

일차배양 (Primary culture) 신선군, 실험군 ( 세포배양액, 우유, 생리식염수각각 2, 4, 7 일군 ) 에서일차배양성공시치수조직으로부터자라나온세포는방추형이거나섬유모세포와유사한형태를보였으며, 각각의군사이에차이가없었다. 양성대조군인신선군은 30개모두일차배양에성공하였고, 음성대조군인건조군은모두일차배양에실패했다.

3 유치치수조직의일차배양성공시에는일주일내에전형적인방추형의섬유모세포형태의세포가자라나오는것이관찰되었으며 (Fig. 2A), 배양실패시에는일주일이지난후에도치수조직으로부터자라서나오는세포가관찰되지않았다 (Fig. 2B). 3. 통계분석 (Statistical analysis) 결과분석은 SPSS (19.0, IBM, Chicago, IL, USA) 프로그램을사용하였고치수세포일차배양성공률은 fisher s exact test (* : p < 0.05) 를시행하였다. Ⅲ. 결과 1. 일차배양 (Primary culture) 신선군, 실험군 ( 세포배양액, 우유, 생리식염수각각 2, 4, 7 일군 ) 에서일차배양성공시치수조직으로부터자라나온세포는방추형이거나섬유모세포와유사한형태를보였으며, 각각의군사이에차이가없었다. 양성대조군인신선군은 30개모두일차배양에성공하였고, 음성대조군인건조군은모두일차배양에실패했다. 세포배양액, 우유, 생리식염수실험군중 2일보관군은모두 90% 이상의일차배양성공률을보였고, 4일보관군도세포배양액과우유에서 90%, 생리식염수에서 86.7% 의높은성공률을보였다. 7일보관군은우유와생리식염수에서급격한성공률의감소가관찰되었고 (Table 1), 세포배양액과비교시통계적으로유의한차이를나타냈다 (Fig. 3). Fig. 2. Primary culture from deciduous teeth. (A) Pulpal cell outgrowth from pulp explants of fresh teeth. (B) Failure of primary culture. Scale bars: 100 μm in A and B. Fig. 3. The success of primary culture within pulp tissues of deciduous teeth conserved in cell culture medium (n=30), milk (n=30) and saline (n=30) for 2, 4 and 7days, respectively. Fisher s exact test (* : p < 0.05). Table 1. The success rate of primary culture from deciduous pulp tissues from fresh teeth, dried teeth and teeth conserved in cell culture medium, milk and saline for 2, 4 and 7days, respectively Fresh Dry Cell culture medium Milk Saline Days Success Failure Total Success rate (%)

4 Ⅳ. 총괄및고찰줄기세포란분화되지않은단일세포로서자가재생능력, 성숙된전구세포생산능력및최종목적세포로의분화능력을가진세포이다. 간엽성줄기세포는골수조직, 지방조직, 근육조직, 혈액조직, 피부조직및치아조직등 13) 에서추출가능하며, 최소한세가지특징을가지는데, 첫번째는배양판바닥에붙어서배양되며, 두번째는 CD105, CD73, CD90과같은세포표면표시자를발현하고, 세번째는특정분화조건에서골모세포, 지방세포, 연골모세포로분화가가능한점이다 14). 치아의치수조직에서배양된세포는자가재생능력, 다양한조직으로의분화능력및자가복제능력을가져골수유래간엽성줄기세포와유사한특성을가진다 15). 본연구와같은방법인 outgrowth method 로치수조직을배양하여얻은세포에서배아줄기세포의표지자인 Oct-4와 Nanog, 외배엽성중간엽줄기세포표지자인 Nestin, 혈관주위세포나신경다발주위세포에서발견되는초기간엽성줄기세포표지자인Stro-1 16) 및간엽성줄기세포표지자로알려진 CD146 17) 이발현되었고지방조직과경조직으로의분화가능성을확인하였다 3). 본연구에서배양한세포가줄기세포의특징을갖는지추가적인실험을통한평가와확인이반드시필요하다. 조직으로부터세포를일차배양하는방법에는 enzyme digestion, mechanical disaggregation, 그리고 outgrowth method (explants culture법 ) 등 18) 이있다. Outgrowth method는조직을잘게자른다음배양접시에고정시킨후세포가자라나오도록하는방법으로세포가자라나오기까지시간은많이걸리나균일한세포군을얻을수있고조직이작은경우에유리하며비용을줄일수있는점이장점이있다 19). 이전까지대부분의유치세포의배양은 enzyme digestion을이용하였으며 1,20-22), outgrowth method 23) 를이용한연구는일부보고되었다. 치근흡수가진행되어자연탈락시기의유치치수에서얻을수있는조직의양이적어본연구에서는 outgrowth method가유리할것으로판단되었다 3). 지금까지발거된유치를저장용액에보관하는것에대한연구는거의없었기때문에영구치의이식이나재식을위한재식치아주변인대세포및치주인대세포에서얻은섬유모세포의생존과관련된연구를살펴보면, 세포의생존에영향을미치는요인으로는온도, 필수영양소함유, 세균과독소의유무, 건조상태, 삼투압, ph 등 9,24) 이있다. 본연구에서발거된유치는 4 의저장용액에보관했는데이유는비교적장기간의보관이필요한실험의목적상보관온도가낮을수록보관용액이부패하거나세균이번식할가능성이적으며세포의부종이감소하고세포의생존력이상승되기때문이다 25). Souza 등 10) 의연구에따르면치주인대조직을배양하여얻은세포를 5 와 20 우유에서보관한결과, 120시간부터 5 에보관한치주인대세포의생존율이유의하게더높게나타났다고보고하였다. 본실험에서세포배양액, 우유, 생리식염수가보관용액으로 사용되었다. 세포배양액에는세포의생존에필요한영양소, 항생제및항진균제가포함되어유리한환경을제공하며, 우유는 Ca 2+, Mg 2+, glucose, platelet derived growth factor (PDGF) 등을포함하고있어서세포보호효과를갖는다 12). 각보관용액에대한이전의연구결과를살펴보면 Huang 등 11) 은치주인대조직을배양하여얻은세포에서생리식염수는 3시간, 우유는 72시간까지생존력유지가가능하다고보고했다. Souza 등 8) 에따르면치주인대조직을배양하여얻은섬유모세포를 sterile Hank s balanced salt solution (shbss), nonsterile HBSS (nhbss), skimmed milk, Save-A-Tooth, Minimum Essential Medium (MEM) 에보관한후생존을관찰한연구에서 37 에서 24시간, 20 에서 48시간까지 skimmed milk가가장좋은결과를나타냈고, 37 에서 48시간이후부터 MEM에서보관한세포의생존률이가장높았다. 따라서치주인대조직을배양하여얻은세포를장기간보관한실험에서는 MEM > 우유 > 생리식염수의결과를보이고있음을알수있다. 치주인대조직을배양하여얻은세포를대상으로한연구방법에차이가있지만본실험에서도 MEM > 우유 > 생리식염수의유사한결과를나타냈다. 세포생존력을유지할수있는기간에대해서는 3시간 26), 6시간 27), 24시간 28), 48시간 8), 72시간 11) 등여러가지실험결과들이보고되었다. Souza 등 10) 은우유와 MEM 용액 ( 양성대조군 ) 에 24시간, 48시간, 72시간, 96시간, 120시간보관시치주인대조직을배양하여얻은세포의생존을비교하였는데, 48시간부터양성대조군의생존율이유의하게높게나타났고, 96시간까지생존율의큰변화가없다가, 120시간이되자우유실험군의생존율이절반으로떨어지는결과가관찰되었다. 치주인대조직을배양하여얻은세포를대상으로한연구방법과는차이가있지만본실험에서도유사한결과를보였는데 4 의세포배양액, 우유및생리식염수에서 2일, 4일간보관한실험군간의세포배양성공률에유의한차이가없었고, 7일군에서절반정도의성공률을나타냈다. 본실험에서 4일과 7일사이의세포의생존률이급격히떨어지는지점에대한연구가필요하다고생각된다. 유치를보관용액에서장기간보관하는과정과배양하는과정에서미생물에의한오염가능성이있으며, 이에의한영향을최소화하기위하여 2일마다우유와식염수를교체하여주었고, 세포배양액내 streptomycin 농도를 300 μg/ml로조절하였고, 항진균제인 amphotericin B를 2.5 μg/ml 추가하였다. 본연구는치수조직세포의활성도를관찰하거나배양된세포의수나양을정량화하지못하였고, 단지세포의생존여부만을관찰하였다는한계점이있지만, 가정에서불시에발거된유치를주변에서쉽게구할수있는우유나생리식염수등의저장용액에임시로보관한후줄기세포의공급원으로사용할수있는가능성을제시하여지금보다더많은유치를줄기세포연구에이용할수있게해준다는점에서그의의를찾을수있다. 4

5 Ⅴ. 결론 4 냉장보관시세포배양액, 우유그리고생리식염수에서보관시 4일까지높은세포배양성공률을보이며세가지저장용액의종류에따른유의한차이가없다. 유치발거후우유나생리식염수등의보관용액에일정기간보관후그치수조직을세포배양에사용하는것이가능하지만보관기간이길어질수록세포획득가능성이줄어든다. References 1. Miura M, Gronthos S, Zhao M, et al.: SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A, 100: , Karaoz E, Dogan BN, Aksoy A, et al.: Isolation and in vitro characterisation of dental pulp stem cells from natal teeth. Histochem Cell Biol, 133:95-112, Song JS, Kim Sh, Kim SO, et al.: Characterization of Stem Cells Obtained from the Dental Pulp and Periodontal Ligament of Deciduous Teeth. Tissue Engineering and Regenerative Medicine, 7, Wang X, Sha XJ, Li GH, et al.: Comparative characterization of stem cells from human exfoliated deciduous teeth and dental pulp stem cells. Arch Oral Biol, 57: , Kerkis I, Caplan AI: Stem cells in dental pulp of deciduous teeth. Tissue Eng Part B Rev, 18: , Song JS, Kim SO, Kim SH, et al.: In vitro and in vivo characteristics of stem cells derived from the periodontal ligament of human deciduous and permanent teeth. Tissue Eng Part A, 18: , Arora V, Arora P, Munshi AK: Banking stem cells from human exfoliated deciduous teeth (SHED): saving for the future. J Clin Pediatr Dent, 33: , Souza BD, Luckemeyer DD, Felippe WT, et al.: Effect of temperature and storage media on human periodontal ligament fibroblast viability. Dent Traumatol, 26: , Lin DG, Kenny DJ, Barrett EJ, et al.: Storage conditions of avulsed teeth affect the phenotype of cultured human periodontal ligament cells. J Periodontal Res, 35:42-50, de Souza BD, Luckemeyer DD, Felippe WT, et al.: Effect of milk renewal on human periodontal ligament fibroblast viability in vitro. Dent Traumatol, 28: , Huang SC, Remeikis NA, Daniel JC: Effects of longterm exposure of human periodontal ligament cells to milk and other solutions. J Endod, 22:30-33, Alacam T, Gorgul G, Omurlu H, Can M: Lactate dehydrogenase activity in periodontal ligament cells stored in different transport media. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 82: , Liu ZJ, Zhuge Y, Velazquez OC: Trafficking and differentiation of mesenchymal stem cells. J Cell Biochem, 106: , Dominici M, Le Blanc K, Mueller I, et al.: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8: , Gronthos S, Brahim J, Li W, et al.: Stem cell properties of human dental pulp stem cells. J Dent Res, 81: , Shi S, Gronthos S: Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res, 18: , Zannettino AC, Paton S, Arthur A, et al.: Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo. J Cell Physiol, 214: , Freshney R: Culture of animal cells : a manual of basic technique. 5th ed. edn Huang GT, Sonoyama W, Chen J, Park SH: In vitro characterization of human dental pulp cells: various isolation methods and culturing environments. Cell Tissue Res, 324: , Nakamura S, Yamada Y, Katagiri W, et al.: Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. J Endod, 35: , Eslaminejad MB, Vahabi S, Shariati M, Nazarian H: In vitro Growth and Characterization of Stem Cells from Human Dental Pulp of Deciduous Versus Permanent Teeth. J Dent (Tehran), 7: , Govindasamy V, Abdullah AN, Ronald VS, et al.: Inherent differential propensity of dental pulp stem cells derived from human deciduous and permanent 5

6 teeth. J Endod, 36: , Spath L, Rotilio V, Alessandrini M, et al.: Explantderived human dental pulp stem cells enhance differentiation and proliferation potentials. J Cell Mol Med, 14: , Lekic PC, Kenny DJ, Barrett EJ: The influence of storage conditions on the clonogenic capacity of periodontal ligament cells: implications for tooth replantation. Int Endod J, 31: , Jo JH, Kim SO, Choi HJ, et al.: A comparative study of preserving ability of human periodontal ligament cells stored in different temperatured storage media. J Korean Acad Pediatr Dent 34:36-42, Blomlof L, Otteskog P: Viability of human periodontal ligament cells after storage in milk or saliva. Scand J Dent Res, 88: , Lindskog S, Blomlof L, Hammarstrom L: Mitoses and microorganisms in the periodontal membrane after storage in milk or saliva. Scand J Dent Res, 91: , Ashkenazi M, Sarnat H, Keila S: In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in six different media. Endod Dent Traumatol, 15: ,

7 국문초록 탈락유치내치수세포의보관용액과기간에따른생존 박지원 1 송제선 1,2 이제호 1,2 김성오 1,2 전미정 2 정한성 3 손흥규 1,2 1 연세대학교치과대학소아치과학교실, 2 연세대학교치과대학구강과학연구소, 3 연세대학교치과대학구강생물학교실내해부발생학연구실 가정에서유치가탈락하였을경우쉽게구할수있는저장용액에보관한후, 보관되었던유치에서세포를배양할수있다면지금보다더많은유치로부터줄기세포를추출할수있을것이다. 본연구는유치의치수세포가다양한보관용액의종류와기간에영향을받는지확인해보고자하였다. 330개의탈락유치를신선군, 건조군, 세포배양액 2, 4, 7일보관군, 우유 2, 4, 7일보관군, 생리식염수 2, 4, 7일보관군으로각각 30개씩무작위로나누었다. 각군간의유치치수조직세포의일차배양을시행하여성공한개수와실패한개수를계산하여생존성공률을비교하였다. 일차배양결과보관기간이늘어날수록일차배양시세포의생존성공률이낮아지는것으로나타났다. 2일보관군과 4일보관군까지세포배양액, 우유, 생리식염수간의보관용액에따른성공률의유의할만한차이는없었다. 그러나 7일보관군에서는세포배양액에비해우유와생리식염수에보관한유치에서성공률이유의하게떨어지는것으로나타났다. 유치발거후우유나생리식염수등의보관용액에일정기간보관후그치수조직을세포배양에사용하는것이가능하지만보관기간이길어질수록세포획득가능성이줄어든다. 주요어 : 유치보관용액, 치수조직, 세포생존, 우유, 생리식염수 7

DBPIA-NURIMEDIA

DBPIA-NURIMEDIA The e-business Studies Volume 17, Number 4, August, 30, 2016:319~332 Received: 2016/07/28, Accepted: 2016/08/28 Revised: 2016/08/27, Published: 2016/08/30 [ABSTRACT] This paper examined what determina