급속냉동된 쥐 치아의 in vivo MTT 검색법을 이용한 치주인대세포 활성도 평가 = EVALUATION OF PERIODONTAL LIGAMENT CELL VIABILITY IN RAT TEETH AFTER FROZEN PRESERVATION USING IN-VIVO MTT ASSAY

국문 초록 (Abstract)

본 연구의 목적은 흰쥐 상악 대구치를 발거 한 후 급속 냉동보존을 통해 치아를 보관하였을 때 in vivo MTT 검색법을 이용하여 치주인대세포의 활성도를 측정하고자 하였다.
실험 방법은 74마리 4주령의 암컷 Sprague-Dawley계의 흰쥐를 사용하여 각군당 10마리의 쥐에서 상악 좌우 제1, 2대구치를 발거하여 모두 40개의 치아를 사용하였다. 대조군은 즉시 발치군이며 냉동군은 F medium에 5% Dimethylsulfoxide (DMSO) 6% Hydroxyethyl starch (HES)를 포함한 군 (1군), 10% DMSO를 포함한 군 (2군) 그리고 Viaspan에 5% DMSO 6% HES (3군). 10% DMSO를 포함한 군 (4군)으로 나누어 1주일간 액체질소에 냉동한 뒤 해동하였다. 냉장군은 F medium (5군)와 Viaspan(6군)에 넣어 1주간 4℃ 냉장고에서 보관하였다. 냉동 및 냉장보존한 후에는 in vivo MTT 검색법을 시행하였다. 개개 치아의 치근면 단위면적으로 표준화하기 위해 치근을 1% eosin Y 용액에 12시간 담근 후 1% acid alcohol로 용해시켜 530 ㎚에서 측정한 흡광도 값을 in vivo MTT 측정값으로 나누었다. 통계 분석을 위해 Two way ANOVA와 Duncan's Multiple Range Test를 95% 신뢰 구간에서 시행하였다. 그리고 각 군당 2개의 치아를 같은 방법으로 처리한 후 10㎛ 두께로 냉동 절단하여 광학 현미경과 편광 현미경하에서 관찰하였다.
1, 2군은 3, 4군보다 높은 흡광도를 나타내었으며 6군은 5군보다 높은 흡광도를 나타내었다 (p < 0.05). 광학 현미경 관찰에서 MTT 결정은 파란색을, 편광현미경에서는 밝은 오렌지색을 나타내었고 전체적으로 in vivo MTT 검색법의 결과와 같은 양상을 나타내었다.
본 연굴의 결과 5%, 10% DMSO를사용한 F medium 배지로 냉동보존한 경우가 Viaspan을 배지로 냉동하였을 때나 냉장보존 하였을 때보다 통계적으로 유의차 있게 높은 세포 활성도를 나타내었다 (p < 0.05).

다국어 초록 (Multilingual Abstract)

The purpose of this study was to examine the viability of PDL cells in rat molars by using in vivo MTT assay, which was used to compare fast cryopreservation group by liquid nitrogen (-196℃) with 4℃ cold preservation group.
A total of 74 Sprague-Dawley white female rats of 4 week-old with a body weight of 100 grams were used. The maxillary left and right, first and second molars were extracted as atraumatically as possible under ketamine anesthesia.
Ten teeth of each group were divided as six experimental groups depending upon the preservation. Cryopreservation groups were Group 1 (5% DMSO 6% HES in F medium). Group 2 (10% DMSO in F medium). Group 3 (5% DMSO 6% HES in Viaspan). Group 4 (10% DMSO in Viaspan) which were cryopreserved for 1 week and cold preservation groups were Group 5 (F medium), Group 6 (Viaspan) at 4℃ for 1 week. Immediate extraction group was used as a control After preservation and thawing, the in vivo MTT assay was processed. Two way ANOVA and Duncan's Multiple Range Test was performed at the 95% level of confidence. Another 2 teeth of each group were treated as the same manner and frozen sections 10 ㎛ thick for microscopic observation.
The value of optical density obtained after in vivo MTT analysis was divided by the value of eosin staining for tissue volume standardization. Group 1, 2 had significantly higher optical density than Group 3 and 4 which had the lowest OD value. Group 6 had higher OD value than in Group 5 (P < 0.05). Histological findings of periodontal ligament cell. after being stained with MTT solution were consistent with the in vivo MTT assay results.
In this study, the groups which were frozen with DMSO as a cryoprotectant and the groups with F medium showed the best results.

참고문헌 (Reference)

• 1 "vitro viability mitogenicity and clonogenic capacity of periodontal ligament cells after storage in six different media" 15 (15): 149-156, 1999
• 2 "vitro viability mitogenicity and clonogenic capacity of periodontal ligament cells after storage in four media at room temperature" 16 (16): 63-70, 2000
• 3 "Tritiated thymidine uptake in periodontal ligament cells of dogs teeth stored in various media for extended time periods" 13 (13): 223-227, 1997
• 4 "Transplantation tooth from bank" Hiroshima University School of Dentistry 2005
• 5 "Tooth transplantation by teeth bank approach to human Hiroshima Department of Orthodontics" Hiroshima University School of Dentistry 2005
• 6 "The influence of storage conditions on the clonogenic capacity of periodontal ligament cells implications for tooth replantation" 31 (31): 137-140, 1998
• 7 "The history and principles of cryopreservation Semin Reprod Med" 20 (20): 5-13, 2002
• 8 "The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells" 19 (19): 283-287, 1997
• 9 "The effect of limited drying or removal of the periodontal ligament Periodontal healing after replantation of mature permanent incisors in monkeys" 1-13, 1981
• 10 "The diverse actions of dimethyl sulphoxide an indicator of membrane transport activity" 254 139-63 255 65, 1990
• 11 "Sigma-Aldrich Handbook of Stains" 1990
• 12 "Rapid colorimetric assay for cellular growth and survival application to proliferation and cytotoxicity assays" j immu (j immu): 1 55-2 63, 1983
• 13 "Predictability of in vivo chemosensitivity by in vitro MTT assay with reference to the clonogenic assay" 12-8, 1989
• 14 "Periodontal regeneration of transplanted rat molars after cryopreservation" 49 (49): 59-69, 2004
• 15 "Periodontal ligament vitality and histologic healing of teeth stored for extended periods before transplantation" 14 (14): 79-83, 1998
• 16 "Periodontal healing of replanted dog teeth stored in Viaspan" 183-81992
• 17 "Pathology of Wound Healing Clinical Aspect Transplantation and Replantation of Teeth Practical Use of Periodontal Ligament Korean Edition ed" Ishiyaku Publishers Inc 1995
• 18 "Noncolorimetric measurement of cell activity in three-dimensional histoculture using the tetrazolium dye 3- the pixel image analysis of formazan crystals" 5 5 8-13, 1992
• 19 "Mechanism of cryoprotection by extracellular polymeric solutes" 509-18, 1988
• 20 "Islet cryopreservation using intracellular preservation solutions" 10 (10): 583-589, 2001
• 21 "Hypothermic preservation of hepatocytes" 19 (19): 1118-1127, 2003
• 22 "Freezing of living cells" 3-42, 1984
• 23 "Evaluation of periodontal ligament cell viability in rat teeth using MTT assay" Yonsei University 2003
• 24 "Evaluation of periodontal ligament cell viability in rat teeth according to various extra oral dry time using MTT assay and a histologic verification" 2004
• 25 "Effect of storage in media with different ion strengths and osmolalities on human periodontal ligament cells" 180-71981
• 26 "Effect of DMSO exposure without cryopreservation on hematopoietic progenitor cells" Bone Marrow Transplant 11 389-93, 1993
• 27 "Dynamic changes in apoptotic and necrotic cell death correlate with severity of ischemia reperfusion injury in lung transplantation" 162 (162): 1932-1939, 2000
• 28 "Cryopreserved rat liver slices a critical evaluation of cell viability histological integrity and drug metabolizing enzymes" 41 (41): 135-144, 2000
• 29 "Cryopreservation with hydroxyethylstarch HES + dimethylsulfoxide DMSO gives better results than DMSO alone" 91 (91): 97-102, 2004
• 30 "Cryopreservation of teeth before replantation or transplantation Atlas of replantation and transplantation of teeth" 241-56, 1992
• 31 "Cryopreservation of mature teeth before replantation in monkeys Effect of different cryoprotective agents and freezing devices" 425-36, 1983
• 32 "Cryopreservation of isolated rat hepatocytes a critical evaluation of freezing and thawing conditions" 323-25 30, 1988
• 33 "Cryopreservation before replantation of mature teeth in monkeys Effect of preincubation different freezing and equilibration rates and endodontic treatment upon periodontal healing" 350-61, 1985
• 34 "Cryopreservation as long-term storage of teeth for transplantation or replantation" 30-2, 1986
• 35 "Correlation of drug response in human tumors histocultured in vitro with an image-analysis MTT end point and in vivo xenografted in nude mice" 1373-6, 1992
• 36 "Autotransplantation of cryopreserved tooth in connection with orthodontic treatment" 67-72, 1986
• 37 "Atlas of replantation & transplantation of teeth" -256, 1992
• 38 "A two-factor hypothesis of freezing injury Evidence from Chinese hamster tissue-culture cells" 345-55, 1972