Restorative Dentistry and Endodontics

  • 제34권4호
  • /
  • Pages.356-363
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  • 2009
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  • 2234-7658(pISSN)
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  • 2234-7666(eISSN)
대한치과보존학회 (The Korean Academy of Conservative Dentistry)
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압력 저속 냉동 방법의 쥐 치아 치주인대세포 보존 효율 평가

THE EFFICACY OF PROGRAMMED CRYO-PRESERVATION UNDER PRESSURE IN RAT PERIODONTAL LIGAMENT CELLS

  • 이영은 (연세대학교 치과대학 보존학 교실) ;
  • 김의성 (연세대학교 치과대학 보존학 교실) ;
  • 김진 (연세대학교 구강병리학 교실 및 구강종양 연구소) ;
  • 한승훈 (강북삼성병원 보존과) ;
  • 이승종 (연세대학교 치과대학 보존학 교실)
  • Lee, Young-Eun (Department of Conservative Dentistry, College of Dentistry, Yonsei University) ;
  • Kim, Eui-Seong (Department of Conservative Dentistry, College of Dentistry, Yonsei University) ;
  • Kim, Jin (Department of Oral Pathology, Oral Cancer Research Center, Yonsei University) ;
  • Han, Seung-Hoon (Department of Conservative Dentistry, Kangbuk Samsung Hospital) ;
  • Lee, Seung-Jong (Department of Conservative Dentistry, College of Dentistry, Yonsei University)
  • 발행 : 2009.07.31
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초록

본 연구의 목적은 흰 쥐의 상악 대구치를 발거한 후 치주인대세포를 압력을 이용한 저속 냉동 보관법으로 냉동 보관시 치주인대세포의 활성도를 저속 냉동법, 냉장 보관법과 비교하여 평가하고자 하였다. 생후 4주된 암컷 Sprague-Dawley계 흰쥐의 상악 좌우 제 1,2 대구치를 발거하여 각 군 당 18개의 쥐 치아를 MTT, WST-1 검색에 이용하였다. 실험군은 4개군으로 대조군은 즉시 발치군이며, $4^{\circ}C$냉장고에 일주일간 보관한 냉장군, 발치 후 동해방지제 처리과정을 거쳐 $4^{\circ}C$에서 $-35^{\circ}C$까지 $-0.5^{\circ}C$/min 속도로 서서히 냉동시킨 뒤 $-196^{\circ}C$에 냉동한 저속 냉동군, 3MPa의 압력을 가하고 $-0.5^{\circ}C$/min 속도로 $4^{\circ}C$에서 $-35^{\circ}C$까지 서서히 냉동시킨 뒤 $-196^{\circ}C$에 냉동한 압력 저속 냉동군으로 나누었다. 보존액은 F medium을 사용했으며 동해방지제로 10% dimethylsulfoxide(DMSO)를 사용하였다. 일주일 후 해동하여 MTT, WST-1검색을 시행한 뒤 치근면을 단위면적으로 표준화하기 위해 MTT, WST-1 측정값을 Eosin 염색 후 530nm에서 측정한 흡광도 값으로 나누었다. 통계 분석을 위해 one way ANOVA를 시행하였으며 사후 검정으로는 Tukey 방법을 사용하였고 결과는 다음과 같다. 1. MTT 검색에 의한 흡광도를 Eosin 염색 후 측정한 흡광도로 나눈 값에서는 압력 저속 냉동군은 즉시 발치군보다 유의성 있게 세포 활성도가 낮았으나(p<0.05), 저속 냉동군이나 냉장군과 비교할 때는 높은 세포 활성도를 보이며 통계적 유의성이 있었다(p<0.05). 2. WST-1 검색에 의한 흡광도를 Eosin 염색 후 측정한 흡광도로 나눈 값에서도 MTT 검색과 마찬가지로 압력 저속 냉동군에서는 즉시 발치군보다 유의성 있게 세포 활성도가 낮았으나(p<0.05), 저속 냉동군이나 냉장군과 비교할때는 높은 세포 활성도를 보이며 통계적 유의성이 있었다(p<0.05). 위의 결과를 통해, 3MPa(30기압) 압력을 이용한 저속 냉동 보관법은 $4^{\circ}C$냉장법이나 압력을 사용하지 않은 다른 냉동 보관법에 비해 우수한 쥐 치아의 치주인대세포의 활성도를 보여 차후 치아의 재식/이식을 위한 중-장기 치아보관을 위한 방법으로의 가능성을 제시하였다.

The purpose of this study was to evaluate the viability of periodontal ligament cells in rat teeth using slow cryo-preservation method under pressure by means of MTT assay and WST-1 assay. Eighteen teeth of Sprague-Dawley white female rats of 4 week-old were used for each group. Both sides of the first and second maxillary molars were extracted as atraumatically as possible under Tiletamine anesthesia. The experimental groups were group 1 (Immediate control), group 2 (Cold preservation at $4^{\circ}C$for 1 week), group 3 (Slow freezing), group 4 (Slow freezing under pressure of 3 MPa). F-medium and 10% DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups, thawing was performed in $37^{\circ}C$water bath, then MTT assay and WST-1 assay were processed. One way ANOVA and Tukey method were performed at the 95% level of confidence. The values of optical density obtained by MTT assay and WST-1 were divided by the values of eosin staining for tissue volume standardization. In both MTT and WST-1 assay, group 4 showed significantly higher viability of periodontal ligament cells than group 2 and 3 (p < 0.05), but showed lower viability than immediate control group. By the results of this study, slow cryo-preservation method under pressure suggests the possibility for long term cryo-preservation of the teeth.

키워드

참고문헌

  1. Schwartz O, Andreasen JO. Cryopreservation of mature teeth before replantation in monkeys(I). Effect of different cryopreservation agents and freezing devices. Int J Oral Surg 12(6):425-436, 1983 https://doi.org/10.1016/S0300-9785(83)80034-9
  2. Schwartz O, Andreasen JO, Greve T. Cryopreservation before replantation of mature teeth in monkeys(II). Effect of preincubation, different freezing and equilibration rates and endodontic treatment upon periodontal healing. Int J Oral Surg 14(4):350-361, 1985 https://doi.org/10.1016/S0300-9785(85)80025-9
  3. Kristerson L. Autotransplantation of human premolars. A clinical and radiographic study of 100 teeth. Int J Oral Surg 14(2):200-213, 1985 https://doi.org/10.1016/S0300-9785(85)80093-4
  4. Lindskog S, Blomlof L, Hammarstrom L. Repair of periodontal tissues in vivo and in vitro. J Clin Periodontol 10(2):188-205, 1983 https://doi.org/10.1111/j.1600-051X.1983.tb02207.x
  5. Melcher AH. Repair of wounds in the periodontium of the rat. Influence of periodontal ligament on osteogenesis. Arch Oral Biol 15(12):1183-1204, 1970 https://doi.org/10.1016/0003-9969(70)90010-5
  6. Andreasen JO. Interrelation between alveolar bone and periodontal ligament repair after replantation of mature permanent incisors in monkeys. J Periodontal Res 16(2):228-235, 1981 https://doi.org/10.1111/j.1600-0765.1981.tb00970.x
  7. Kawata T. Tooth transplantation by teeth bankapproach to human-Hiroshima. Department of Orthodontics, Hiroshima University School of Dentistry. 2005
  8. Kaku M, Kamata H, Kawata T. Cryopreservation of PDL cells by use of program freezer with magnetic field for tooth banking. Dent Jpn. 43:82-86, 2007
  9. Ahn HJ. Kim ES, Kim J, Kim DW, Kim KY, Lee CY, Lee SJ. Evaluation of viability of periodontal ligament cell in rat teeth-using slow cryoperservation method with magnetic field. J Kor Acad of Cons Dent 33(4):332-340, 2008 https://doi.org/10.5395/JKACD.2008.33.4.332
  10. Kim JW, Kim ES, Kim J, Lee SJ. Evaluation of periodontal ligament cell viability in rat teeth after frozen preservation using in-vivo MTT assay. J Kor Acad of Cons Dent 3(3):192-202, 2006
  11. Kim ES, Jeon IS, Kim JW, Kim J, Juhn HS, Lee SJ. An MTT-based method for quantification of periodontal ligament cell viability. Oral Dis 13(5):495-499, 2007 https://doi.org/10.1111/j.1601-0825.2006.01328.x
  12. Rubinsky B. Principles of low temperature cell preservation. Heart Fail Rev 8(3):277-284, 2003 https://doi.org/10.1023/A:1024734003814
  13. Schluter O, Urrutia Benet G, Heinz V, Knorr D. Metastable states of water and ice during pressuresupported freezing of potato tissue. Biotechnol Prog 20(3):799-810, 2004 https://doi.org/10.1021/bp0340279
  14. Zhu SM, Ramaswamy HS, Le Bail A. Ice crystal formation in gelatin gel during pressure shift versus conventional freezing. J of Food Engineering 66(1):69-76, 2005 https://doi.org/10.1016/j.jfoodeng.2004.02.035
  15. Kawasaki N, Hamamoto Y, Nakajima T, Irie K, Ozawa H. Periodontal regeneration of transplanted rat molars after cryopreservation. Arch Oral Biol 49(1):59-69, 2004 https://doi.org/10.1016/j.archoralbio.2003.07.002
  16. Andreasen JO, Schwartz O. Atlas of replantation and transplantation of teeth. Mediglobe SA, Fribourg, Switzerland. 1992
  17. Ashwood-smith MJ. Low temperature preservation of cells, tissues and organs. Pitman Medicals 19-45, 1980
  18. Farrant J. Water transport and cell survival in cryobiological procedures. Philos Trans R Soc Lond B Biol Sci 278(959):191-205, 1977 https://doi.org/10.1098/rstb.1977.0037
  19. Nei T. Structure and function of frozen cells freezing patterns and post-thaw survival. J Microsc 112(2):197-204, 1978 https://doi.org/10.1111/j.1365-2818.1978.tb01165.x
  20. Shimada K. Effects of cryoprotective additives on intracellular ice formation and survival in very rapidly cooled HeLa cells. Contrib Inst Low Temp Sci Ser 19:49-69, 1977
  21. Mazur P, Leibo SP, Chu EH. A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Exp Cell Res 71(2):345-355, 1972 https://doi.org/10.1016/0014-4827(72)90303-5
  22. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 247:125-142, 1984
  23. Gao D, Critser JK. Mechanisms of cryoinjury in living cells. ILAR J 41(4):187-196, 2000 https://doi.org/10.1093/ilar.41.4.187
  24. Inuzuka K, Unno N, Yamamoto N, Sagara D, Suzuki M, Nishiyama M, Konno H. Effect of hyperbarically oxygenated-perfluorochemical with university of Wisconsin solution on preservation of rat small intestine using an original pressure-resistant portable apparatus. Surgery 142(1):57-66, 2007 https://doi.org/10.1016/j.surg.2007.03.002
  25. Takahashi T, Kakita A, Takahashi Y, I. Sakamoto, K. Yokoyama, T. Fujiu, S. Yamashina, T. Tamaki, Y. Takazawa, R. Muratsubaki. Funtional integrity of the rat liver after subzero preservation under high pressure. Transplant Proc 32(7):1634-1636, 2000 https://doi.org/10.1016/S0041-1345(00)01440-8
  26. Takahashi T, Kakita A, Takahashi Y, Yokoyama K, Sakamoto I, Yamashina S. Preservation of rat livers by supercooling under high pressure. Transplant Proc 33(1-2):916-919, 2001 https://doi.org/10.1016/S0041-1345(00)02268-5
  27. Pribenszky C, Molnar M, Cseh S, Solti L. Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Anim Reprod Sci 87(1-2):143-150, 2005 https://doi.org/10.1016/j.anireprosci.2004.09.007
  28. Pribenszky C, Du Y, Molnar M, Harnos A, Vajta G. Increased stress tolerance of matured pig oocytes after high hydrostatic pressure treatment. Anim Reprod Sci 106(1-2):200-207, 2008 https://doi.org/10.1016/j.anireprosci.2008.01.016
  29. Du Y, Lin L, Schmidt M, Bogh IB, Kragh PM, Sorensen CB, Li J, Purup S, Pribenszky C, Molnar M, Kuwayama M, Zhang X, Yang H, Bolund L, Vajta G. High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival. Cloning Stem Cells 10(3):325-330, 2008 https://doi.org/10.1089/clo.2007.0089
  30. Abe F, Kato C, Horikoshi K. Pressure regulated metabolism in microorganisms. Trends Microbiol 7(11):447-453, 1999 https://doi.org/10.1016/S0966-842X(99)01608-X
  31. Aldridge BE, Bruner LJ. Pressure effects on mechanisms of charge transport across bilayer membranes. Biochim Biophys Acta 817(2):343-354, 1985 https://doi.org/10.1016/0005-2736(85)90037-9
  32. Huang SY, Pribenszky C, Kuo YH., et al. The effect of hydrostatic pressure treatment on the protein profile of boar spermatozoa before and after freezing. Proceedings of the 6th International Conference on Boar Semen Preservation, Alliston, Ontario, Cananda, p.I.-34, 2007
  33. Kaarniranta K, Elo M, Sironen R, Lammi MJ, Goldring MB, Eriksson JE, Sistonen L, Helminen HJ. Hsp 70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci USA 95(5):2319-2324, 1998 https://doi.org/10.1073/pnas.95.5.2319
  34. Elo MA, Sironen RK, Karjalainen HM, Kaarniranta KK, Helminen HJ, Lammi MJ. Specific induction of heat shock protein 90 beta by high hydrostatic pressure. Biorheology 40(1-3):141-146, 2003
  35. Du Y, Pribenszky C, Molnar M, Zhang X, Yang H, Kuwayama M, Pedersen AM, Villemoes K, Bolund L, Vajta G. High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification. Reproduction 135(1):13-17, 2008 https://doi.org/10.1530/REP-07-0362
  36. Fisher S, Maclean AA, Liu M, Cardella JA, Slutsky AS, Suga M, Moreira JF, Keshavjee S. Dynamic changes in apoptotic and necrotic cell death correlate with severity of ischemia-reperfusion injury in lung transplantation. Am J Respir Crit Care Med 162 (5):1932-9, 2000 https://doi.org/10.1164/ajrccm.162.5.9910064
  37. Carlson MA. Technical note: assay of cell quantity in the fibroblast populated collagen matrix with a tetrazolium reagent. Eur Cell Mater 12:44-48, 2006
  38. Alotto D, Ariotti S, Graziano S, Verrua R, Stella M, Magliacani G, Castagnoli C. The role of quality control in a skin bank: tissue viability determination. Cell Tissue Bank 3(1):3-10, 2002 https://doi.org/10.1023/A:1021846703301

피인용 문헌

  1. Comparison of viability of oral epithelial cells stored by different freezing methods vol.34, pp.6, 2009, https://doi.org/10.5395/JKACD.2009.34.6.491
  2. The evaluation of periodontal ligament cells of rat teeth after low-temperature preservation under high pressure vol.35, pp.4, 2010, https://doi.org/10.5395/JKACD.2010.35.4.285
  3. Effects of Slow Programmable Cryopreservation on Preserving Viability of the Cultured Periodontal Ligament Cells from Human Impacted Third Molar vol.8, pp.2, 2015, https://doi.org/10.5856/JKDS.2015.8.2.57