Evaluation of periodontal ligament cell viability in rat teeth after frozen preservation using in-vivo MTT assay

Jae-Wook Kim; Eui-Sung Kim; Jin Kim; Seung-Jong Lee

doi:10.5395/JKACD.2006.31.3.192

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Kim, Kim, Kim, and Lee: Evaluation of periodontal ligament cell viability in rat teeth after frozen preservation using in-vivo MTT assay

Original Article

Journal of Korean Academy of Conservative Dentistry

Journal of Korean Academy of Conservative Dentistry 2006; 31(3): 192-202.

Published online: 31 May 2006

DOI: https://doi.org/10.5395/JKACD.2006.31.3.192

Evaluation of periodontal ligament cell viability in rat teeth after frozen preservation using in-vivo MTT assay

Jae-Wook Kim, Eui-Sung Kim, Jin Kim, Seung-Jong Lee

Department of Conservative Dentistry, College of Dentistry, Yonsei University, Seoul, Korea.

Corresponding Author: Seung-Jong Lee. Department of Conservative Dentistry, College of Dentistry, Yonsei University, 134 Shinchon-Dong, Seodaemun-Ku, Seoul, Korea, 120-752. Tel: 82-2-361-8700, Fax: 82-2-313-7575, sjlee@yumc.yonsei.ac.kr

Received 19 January 2006 Revised 20 March 2006 Accepted 21 March 2006

(open-access):

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 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 µm 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.

Keywords: Periodontal ligament cell, Vitality, In-vivo MTT assay, Cryopreservation, Cold preservation, Optical density

Figures and Tables

Figure 1

96 well plate after in vivo MTT assay.

Figure 2

Optical density of MTT, Eosin stain and MTT/Eosin following different experimental groups in Mx 1st and Mx 2nd molar.

A, B, C, D, a, b, c, d mean grouping by Duncan's Multiple Range Test

Means with the same letter are not significantly different. P < 0.05

Control; immediate extraction.

Group 1; cryopreservation with 5% DMSO and 6% HES in F medium for 1 week.

Group 2; cryopreservation with 10% DMSO in F medium for 1 week.

Group 3; cryopreservation with 5% DMSO and 6% HES in Viaspan® for 1 week.

Group 4; cryopreservation with 10% DMSO in Viaspan® for 1 week.

Group 5; cold preservation in F medium at 4℃ for 1 week.

Group 6; cold preservation in Viaspan® at 4℃ for 1 week.

Figure 3

Immediate control with MTT stain, optical and polarized microscopic view.

Figure 4

Cryopreservation with 5% DMSO and 6% HES in F medium group with MTT stain, optical and polarized microscopic view.

Figure 5

Cryopreservation with 10% DMSO in F medium group with MTT stain, optical and polarized microscopic view.

Figure 6

Cryopreservation with 5% DMSO and 6% HES in Viaspan® group with MTT stain, optical and polarized microscopic view.

Figure 7

Cryopreservation with 10% DMSO in Viaspan® group with MTT stain, optical and polarized microscopic view.

Figure 8

Cold preservation in F medium at 4℃ group with MTT stain, optical and polarized microscopic view.

Figure 9

Cold preservation in Viaspan® at 4℃ group with MTT stain, optical and polarized microscopic view.

Table 1

Experimental groups

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