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

Articles

Page Path: HOME > Restor Dent Endod > Volume 31(3); 2006 > Article

Original Article 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; Journal of Korean Academy of Conservative Dentistry 2006;31(3):192-202.
DOI: https://doi.org/10.5395/JKACD.2006.31.3.192
Published online: May 31, 2006

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: January 19, 2006 • Revised: March 20, 2006 • Accepted: March 21, 2006

21 Views
0 Download

prev next

Full Article

Download PDF

Abstract
REFERENCES

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

REFERENCES

1. Andreasen JO, Kristerson L. The effect of limited drying or removal of the periodontal ligament. Periodontal healing after replantation of mature permanent incisors in monkeys. Acta Odontol Scand. 1981;39(1):1-13.PubMed
2. Schwartz O, Andreasen JO. Cryopreservation of mature teeth before replantation in monkeys (I). Effect of different cryoprotective agents and freezing devices. Int J Oral Surg. 1983;12(6):425-436.PubMed
3. Schwartz O, Andreasen JO, Greve T. Cryopreservation before replantation of mature teeth in monkeys. (II). Effect of preincubation, different freezing and equilibration rates and endodontic treatment upon periodontal healing. Int J Oral Surg. 1985;14(4):350-361.PubMed
4. Schwartz O. Cryopreservation as long-term storage of teeth for transplantation or replantation. Int J Oral Maxillofac Surg. 1986;15(1):30-32.Article PubMed
5. Pegg DE. The history and principles of cryopreservation. Semin Reprod Med. 2002;20(1):5-13.Article PubMed
6. Neulieb RL, Neulieb MK. The diverse actions of dimethyl sulphoxide: an indicator of membrane transport activity. Cytobios. 1990;63(254-255):139-165.PubMed
7. Katayama Y, Yano T, Bessho A, Deguchi S, Sunami K, Mahmut N. The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells. Bone Marrow Transplant. 1997;19(3):283-287.Article PubMed PDF
8. Schwartz O. Cryopreservation of teeth before replantation or transplantation. Atlas of replantation and transplantation of teeth. 1992;Medglobe; 241-256.
9. Schwartz O, Rank CP. Autotransplantation of cryopreserved tooth in connection with orthodontic treatment. Am J Orthod Dentofacial Orthop. 1986;90(1):67-72.Article PubMed
10. Hupp JG, Mesaros SV, Aukhil I, Trope M. Periodontal ligament vitality and histologic healing of teeth stored for extended periods before transplantation. Endod Dent Traumatol. 1998;14(2):79-83.Article PubMed
11. Lekic PC, Kenny DJ, Barrett EJ. The influence of storage conditions on the clonogenic capacity of periodontal ligament cells: implications for tooth replantation. Int Endod J. 1998;31(2):137-140.PubMed
12. Ashkenazi M, Sarnat H, Keila S. In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in six different media. Endod Dent Traumatol. 1999;15(4):149-156.Article PubMed
13. Hupp JG, Trope M, Mesaros SV, Aukhil I. Tritiated thymidine uptake in periodontal ligament cells of dogs' teeth stored in various media for extended time periods. Endod Dent Traumatol. 1997;13(5):223-237.Article PubMed
14. Ashkenazi M, Marouni M, Sarnat H. In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in four media at room temperature. Endod Dent Traumatol. 2000;16(2):63-70.Article PubMed PDF
15. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63.PubMed
16. Shimoyama Y, Kubota T, Watanabe M, Ishibiki K, Abe O. Predictability of in vivo chemosensitivity by in vitro MTT assay with reference to the clonogenic assay. J Surg Oncol. 1989;41(1):12-18.Article PubMed
17. Colangelo D, Guo HY, Connors KM, Silvestro L, Hoffman RM. Noncolorimetric measurement of cell activity in three-dimensional histoculture using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide: the pixel image analysis of formazan crystals. Anal Biochem. 1992;205(1):8-13.Article PubMed
18. Colangelo D, Guo HY, Connors KM, Kubota T, Silvestro L, Hoffman RM. Correlation of drug response in human tumors histocultured in vitro with an imageanalysis MTT end point and in vivo xenografted in nude mice. Anticancer Res. 1992;12(5):1373-1376.PubMed
19. Blomlof L, Otteskog P, Hammarstrom L. Effect of storage in media with different ion strengths and osmolalities on human periodontal ligament cells. Scand J Dent Res. 1981;89(2):180-187.PubMed
20. Trope M, Friedman S. Periodontal healing of replanted dog teeth stored in Viaspan, milk and Hank's balanced salt solution. Endod Dent Traumatol. 1992;8(5):183-188.Article PubMed
21. Kim ES. Evaluation of periodontal ligament cell viability in rat teeth using MTT assay [PhD thesis]. 2003;Yonsei University.
22. Andreasen J. Atlas of replantation & transplantation of teeth. 1992;Mediglobe SA; 242-256.
23. Mazur P, Leibo SP, Chu EH. A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Exp Cell Res. 1972;71(2):345-355.PubMed
24. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol. 1984;247(3 Pt 1):C125-C142.Article PubMed
25. Clapisson G, Salinas C, Malacher P, Michallet M, Philip I, Philip T. Cryopreservation with hydroxyethylstarch (HES) + dimethylsulfoxide (DMSO) gives better results than DMSO alone. Bull Cancer. 2004;91(4):E97-E102.PubMed
26. Kawasaki N, Hamamoto Y, Nakajima T, Irie K, Ozawa H. Periodontal regeneration of transplanted rat molars after cryopreservation. Arch Oral Biol. 2004;49(1):59-69.Article PubMed
27. Rowley SD, Anderson GL. Effect of DMSO exposure without cryopreservation on hematopoietic progenitor cells. Bone Marrow Transplant. 1993;11(5):389-393.PubMed
28. Takahashi T, Hirsh A, Erbe E, Williams RJ. Mechanism of cryoprotection by extracellular polymeric solutes. Biophys J. 1988;54(3):509-518.Article PubMed PMC
29. Martin H, Bournique B, Sarsat JP, Albaladejo V, Lerche-Langrand C. Cryopreserved rat liver slices: a critical evaluation of cell viability, histological integrity, and drug-metabolizing enzymes. Cryobiology. 2000;41(2):135-144.PubMed
30. Lakey JR, Rajotte RV, Fedorow CA, Taylor MJ. Islet cryopreservation using intracellular preservation solutions. Cell Transplant. 2001;10(7):583-589.Article PubMed PDF
31. Chesne C, Guillouzo A. Cryopreservation of isolated rat hepatocytes: a critical evaluation of freezing and thawing conditions. Cryobiology. 1988;25(4):323-330.Article PubMed
32. Fischer S, Maclean AA, Liu M, Cardella JA, Slutsky AS, Suga M. Dynamic changes in apoptotic and necrotic cell death correlate with severity of ischemia-reperfusion injury in lung transplantation. Am J Respir Crit Care Med. 2000;162(5):1932-1939.Article PubMed
33. Meng Q. Hypothermic preservation of hepatocytes. Biotechnol Prog. 2003;19(4):1118-1127.PubMed
34. Shimono M, Lijima K. Pathology of Wound Healing <Clinical Aspect> Transplantation and Replantation of Teeth-Practical Use of Periodontal Ligament-. 1995;Korean Edition. Ishiyaku Publishers, Inc.
35. Jeon IS. Evaluation of periodontal ligament cell viability in rat teeth according to various extra-oral dry time using MTT assay and a histologic verification [PhD. thesis]. 2004;Yonsei university.
36. Green FJ. Sigma-Aldrich Handbook of Stains, Dyes and Indicators: Sigma-aldrich. 1990.
37. Kawata T. Tooth transplantation by teeth bank-approach to human-. 2005;Hiroshima: Department of Orthodontics, Hiroshima University School of Dentistry.
38. Kawata T. Transplantation tooth from bank. 2005;Hiroshima: Department of Orthodontics, Hiroshima University School of Dentistry.

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

Tables & Figures

REFERENCES

Citations

Citations to this article as recorded by

ePub Link

Cite

CITE: export Copy Download Format; Close

Download Citation

Download a citation file in RIS format that can be imported by all major citation management software, including EndNote, ProCite, RefWorks, and Reference Manager.

Format:

RIS — For EndNote, ProCite, RefWorks, and most other reference management software
BibTeX — For JabRef, BibDesk, and other BibTeX-specific software

Include:

Citation for the content below
Citation and abstract for the content below

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

J Korean Acad Conserv Dent. 2006;31(3):192-202. Published online May 31, 2006

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

XML Download

Figure

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

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.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

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

Table 1

Experimental groups

Table 1 Experimental groups