Restorative Dentistry and Endodontics

The Korean Academy of Conservative Dentistry (대한치과보존학회)
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THE EVALUATION OF PERIODONTAL LIGAMENT CELLS OF RAT TEETH AFTER LOW-TEMPERATURE PRESERVATION UNDER HIGH PRESSURE

고압-저온 보관에 따른 쥐 치아 치주인대세포의 활성도 평가

  • Chung, Jin-Ho (Department of Conservative Dentistry, College of Dentistry, Yonsei University) ;
  • Kim, Jin (Department of Oral Pathology, College of Dentistry, Yonsei University) ;
  • Choi, Seong-Ho (Department of Periodontology, College of Dentistry, Yonsei University) ;
  • Kim, Eui-Seong (Department of Conservative Dentistry, College of Dentistry, Yonsei University) ;
  • Park, Ji-Yong (Department of Biotechnology, College of Life Science and Biothchnology, Yonsei University) ;
  • Lee, Seung-Jong (Department of Conservative Dentistry, College of Dentistry, Yonsei University)
  • 정진호 (연세대학교 치과대학 치과보존학교실) ;
  • 김진 (연세대학교 치과대학 구강병리학교실) ;
  • 최성호 (연세대학교 치과대학 치주과학교실) ;
  • 김의성 (연세대학교 치과대학 치과보존학교실) ;
  • 박지용 (연세대학교 생명시스템대학 생명공학과) ;
  • 이승종 (연세대학교 치과대학 치과보존학교실)
  • Received : 2010.05.29
  • Accepted : 2010.07.03
  • Published : 2010.07.30
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Abstract

The purpose of this study was to evaluate the viability of periodontal ligament cells of rat teeth after low-temperature 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 $0^{\circ}C$ under pressure of 2 MPa), group 8 (low-temperature preservation at $0^{\circ}C$ under no additional pressure), group 9 (low-temperature preservation at $-5^{\circ}C$ 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 $37^{\circ}C$ 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 ($0^{\circ}C$/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 $0^{\circ}C$ under pressure of 2 MPa suggest the possibility for long term preservation of teeth.

본 연구의 목적은 흰 쥐의 상악 대구치를 발거한 후 치주인대세포를 $0^{\circ}C$/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), $-5^{\circ}C$/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-1 검색에 이용하였다. 실험군은 9개군으로 대조군은 즉시 발치군이며, 각각 3 MPa, 2 MPa, No Additional Pressure (NAP)의 압력을 가한 후 $4^{\circ}C$에서 $-35^{\circ}C$까지 $-0.5^{\circ}C$/min 속도로 서서히 냉동시킨 뒤 $-196^{\circ}C$에 냉동한 저속 냉동군, 발치 후 동해방지제 처리과정을 거쳐 각각 2 MPa, NP의 압력을 가한 후, $-196^{\circ}C$의 액화질소에 넣어 냉동한 급속 냉동군, 발치 후 각각 2MPa,NP의 압력을 가한 후, $0^{\circ}C$에 보관한 저온 보존군, $-5^{\circ}C$/90 MPa의 초고압 저온 보존군으로 나누었다. 보존액은 F medium을 사용했으며 동해방지제로 10% dimethylsulfoxide (DMSO)를 사용하였다. 치근면을 단위면적으로 표준화하기 위해 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 $0^{\circ}C$/2 MPa 고압 저온 보존군이 즉시 발치군보다 세포 활성도가 낮았으나 통계적 유의차는 없었으며, 저속 압력 냉동군(NP, 2 MPa, 3 MPa)과, 급속 압력 냉동군(NP, 2 MPa), 저온보존군($0^{\circ}C$/NP), 초고압 저온 보존군($-5^{\circ}C$/90 MPa)보다 통계적으로 유의차있게 높은 세포 활성도를 나타내었다(p < 0.05). 2. MTT검색법 및 WST-1 검색법 결과 $-5^{\circ}C$/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, $0^{\circ}C$/2 MPa (20기압)의 고압-저온 보존법이 다른 급속 냉동 보관법(2 MPa, NAP)이나 저속냉동보관법(3, 2 MPa, NAP), $-5^{\circ}C$/90 MPa 초고압 저온 보존법에 비해 우수한 쥐 치아의 치주인대세포의 활성도를 보여 차후 치아의 재이식시 치아보관을 위한 방법으로의 가능성을 제시하였다.

Keywords

References

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