J Korean Acad Conserv Dent.  2006 May;31(3):192-202. 10.5395/JKACD.2006.31.3.192.

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

• Kim JW
• Kim ES
• Kim J
• Lee SJ

• KMID: 2175819
• DOI: http://doi.org/10.5395/JKACD.2006.31.3.192

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 (-196degrees C) with 4degrees C 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(R)), Group 4 (10% DMSO in Viaspan(R)) which were cryopreserved for 1 week and cold preservation groups were Group 5 (F medium), Group 6 (Viaspan(R)) at 4degrees C 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 microm 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.

Keyword

Periodontal ligament cell; Vitality; In-vivo MTT assay; Cryopreservation; Cold preservation; Optical density

MeSH Terms

Anesthesia
Animals
Body Weight
Cell Survival*
Cryopreservation
Dimethyl Sulfoxide
Eosine Yellowish-(YS)
Female
Frozen Sections
Humans
Ketamine
Molar
Nitrogen
Periodontal Ligament*
Rats*
Rats, Sprague-Dawley
Tooth*
Dimethyl Sulfoxide
Eosine Yellowish-(YS)
Ketamine
Nitrogen

Figure

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  Figure 1 96 well plate after in vivo MTT assay.

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  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.

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  Figure 3 Immediate control with MTT stain, optical and polarized microscopic view.

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  Figure 4 Cryopreservation with 5% DMSO and 6% HES in F medium group with MTT stain, optical and polarized microscopic view.

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  Figure 5 Cryopreservation with 10% DMSO in F medium group with MTT stain, optical and polarized microscopic view.

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

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  Figure 7 Cryopreservation with 10% DMSO in Viaspan® group with MTT stain, optical and polarized microscopic view.

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  Figure 8 Cold preservation in F medium at 4℃ group with MTT stain, optical and polarized microscopic view.

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  Figure 9 Cold preservation in Viaspan® at 4℃ group with MTT stain, optical and polarized microscopic view.

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