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고압-저온 보관에 따른 쥐 치아 치주인대세포의 활성도 평가

THE EVALUATION OF PERIODONTAL LIGAMENT CELLS OF RAT TEETH AFTER LOW-TEMPERATURE PRESERVATION UNDER HIGH PRESSURE

이승종|박지용|최성호|김진|김의성|정진호
대한치과보존학회 (Korean Academy Of Conservative Dentistry)|2010.07|Restorative Dentistry and Endodontics|제35권 제4호|pp. 285-294

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MTT2010Viability치주인대세포Cryopreservation활성도Number 4WST-1Rapid freezingPeriodontal ligament cellLow temperature preservation under pressure고압 저온 보존급속 냉동압력 저속 냉동Basic Research Chung JH et al. JKACD Volume 35
주제분류치의학

초록

본 연구의 목적은 흰 쥐의 상악 대구치를 발거한 후 치주인대세포를 0℃/2 MPa고압-저온하에 1주간 보관시켜 MTT,WST-1 검색법을 이용하여 측정한 치주인대세포의 활성도를 저속 냉동법(No Additional Pressure, 2, 3 MPa), 급속 냉동법(No Additional Pressure, 2 MPa), -5℃/90 MPa초고압 저온보존법과 비교하여 평가하는 것이다. 생후 4주된 암컷 Sprague-Dawley계 흰쥐의 상악 좌우 제1, 2대구치를 발거하여 각 군 당 12개의 쥐 치아를 MTT, WST-1검색에 이용하였다. 실험군은 9개군으로 대조군은 즉시 발치군이며, 각각 3 MPa, 2 MPa, No Additional Pressure (NAP)의 압력을 가한 후 4℃에서 -35℃까지 -0.5℃/min 속도로 서서히 냉동시킨 뒤 -196℃에 냉동한 저속 냉동군, 발치 후 동해방지제 처리과정을 거쳐 각각 2 MPa, NP의 압력을 가한 후, -196℃의 액화질소에 넣어 냉동한 급속 냉동군, 발치 후 각각 2MPa,NP의 압력을 가한 후, 0℃에 보관한 저온 보존군, -5℃/90 MPa의 초고압 저온 보존군으로 나누었다. 보존액은 F medium을 사용했으며 동해방지제로 10% dimethylsulfoxide (DMSO)를 사용하였다. 치근면을 단위면적으로 표준화하기위해 MTT, WST-1 측정값을 Eosin 염색 후 530 nm에서 측정한 흡광도 값으로 나누었다. 통계 분석을 위해 one way ANOVA를 시행하였으며 사후 검정으로는 Tukey HSD 방법을 사용하였고 결과는 다음과 같다. 1. MTT 검색법 및 WST-1 검색법 결과 0℃/2 MPa 고압 저온 보존군이 즉시 발치군보다 세포 활성도가 낮았으나 통계적유의차는 없었으며, 저속 압력 냉동군(NP, 2 MPa, 3 MPa)과, 급속 압력 냉동군(NP, 2 MPa), 저온보존군(0℃/NP),초고압 저온 보존군(-5℃/90 MPa)보다 통계적으로 유의차있게 높은 세포 활성도를 나타내었다(p < 0.05). 2. MTT검색법 및 WST-1 검색법 결과 -5℃/90 MPa 초고압 저온 보존군이 가장 낮은 세포 활성도를 나타내었으며, MTT 검사 결과에서는 모든 군에 대해 통계적으로 유의성 있는 결과를 보였다(p < 0.05). 위의 결과를 통해, 0℃/2 MPa (20기압)의 고압-저온 보존법이 다른 급속 냉동 보관법(2 MPa, NAP)이나 저속냉동보관법(3, 2 MPa, NAP), -5℃/90 MPa 초고압 저온 보존법에 비해 우수한 쥐 치아의 치주인대세포의 활성도를 보여 차후 치아의재이식시 치아보관을 위한 방법으로의 가능성을 제시하였다.

Abstract

The purpose of this study was to evaluate the viability of periodontal ligament cells of rat teeth after lowtemperature preservation under high pressure by means of MTT assay, WST-1 assay. 12 teeth of Sprague-Dawley white female rats of 4 week-old were used for each group. Both side of the first and second maxillary molars were extracted as atraumatically as possible under tiletamine anesthesia. The experimental groups were group 1 (Immediate extraction), group 2 (Slow freezing under pressure of 3 MPa), group 3 (Slow freezing under pressure of 2 MPa), group 4 (Slow freezing under no additional pressure), group 5 (Rapid freezing in liquid nitrogen under pressure of 2 MPa), group 6 (Rapid freezing in liquid nitrogen under no additional pressure), group 7 (low-temperature preservation at 0℃ under pressure of 2 MPa), group 8 (low-temperature preservation at 0℃ under no additional pressure),group 9 (low-temperature preservation at -5℃ under pressure of 90 MPa). F-medium and 10%DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups, thawing was performed in 37℃ water bath, then MTT assay, WST-1 assay were processed. One way ANOVA and Tukey HSD 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 7 (0℃/2 MPa) showed higher viability of periodontal ligament cells than other group (2-6, 8) and this was statistically significant (p < 0.05), but showed lower viability than group 1, immediate extraction group (no statistical significance). By the results of this study, low-temperature preservation at 0℃ under pressure of 2 MPa suggest the possibility for long term preservation of teeth. [J Kor Acad Cons Dent 35(4):285-294, 2010]

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