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

Young-Eun Lee; Eui-Seong Kim; Jin Kim; Seung-Hoon Han; Seung-Jong Lee

doi:10.5395/JKACD.2009.34.4.356

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Lee, Kim, Kim, Han, and Lee: THE EFFICACY OF PROGRAMMED CRYO-PRESERVATION UNDER PRESSURE IN RAT PERIODONTAL LIGAMENT CELLS

Original Article

J Korean Acad Conserv Dent 2009;34(4):356-363.

Published online: 14 January 2009

DOI: https://doi.org/10.5395/JKACD.2009.34.4.356

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

Young-Eun Lee¹, Eui-Seong Kim¹, Jin Kim², Seung-Hoon Han³, Seung-Jong Lee^1,^*

¹Department of Conservative Dentistry, College of Dentistry, Yonsei University

²Department of Oral Pathology, Oral Cancer Research Center, Yonsei University

³Department of Conservative Dentistry, Kangbuk Samsung Hospital

^*Corresponding Author: Seung-Jong Lee, Department of Conservative Dentistry, College of Dentistry, Yonsei University, 250 Seongsan-ro Seodaemin-Ku, Seoul, 120-752, Korea, Tel: 82-2-2228-3148 Fax: 82-2-313-7575, E-mail sjlee@yuhs.ac

Received 23 April 2009 Revised 16 June 2009 Accepted 22 June 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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°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°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.

Keywords: periodontal ligament cell, MTT, WST-1, viability, pressure, cryo-preservation

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Figure 1.

Schematic diagram of program freezer with pressure vessel.

Oxygen container : 3MPa of pressure
Program freezer
Pressure bottle
2ml Cryotube: 1ml F medium + 10% DMSO
Tooth treated 10% DMSO
Pressure valve
Thermometer

Table 1.

The averages and standard deviations of optical density of MTT.

Groups	MTT¹	Eosin²	MTT/Eosin³
Immediate control	3.01±0.25	0.49±0.11	6.45±1.46^a
Cold preservation	0.81±0.26	0.42±0.11	2.08±0.94^b
Slow freezing	1.62±0.24	0.47±0.13	3.62±0.88^c
Slow freezing under pressure	1.94±0.44	0.44±0.14	4.71±1.24^d

There were significant differences between groups with different subscripts(p<0.05).

The values of MTT¹ are proportional to the vitality of the cells.

The values of Eosin² are proportional to the area of the periodontal ligament cells.

So, the values of MTT/Eosin³ mean the vitality of the periodontal ligament cells per unit area.

Table 2.

The averages and standard deviations of optical density of WST-1.

Groups	WST-1¹	Eosin²	WST-1/Eosin³
Immediate control	2.31±0.33	0.49±0.13	4.96±1.23^a
Cold preservation	0.58±0.19	0.39±0.12	1.50±0.32^b
Slow freezing	1.11±0.32	0.5±0.20	2.46±1.00^c
Slow freezing under pressure	1.34±0.21	0.41±0.10	3.41±0.81^d

There were significant differences between groups with different subscripts(p<0.05).

The values of WST-1¹ are proportional to the vitality of the cells.

The values of Eosin² are proportional to the area of the periodontal ligament cells.

So the values of WST-1/Eosin³ mean the vitality of the periodontal ligament cells per unit area.

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