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

Article information

Restor Dent Endod. 2006;31(3):192-202

Publication date (electronic) : 2006 May 31

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

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 2006 January 19; Revised 2006 March 20; Accepted 2006 March 21.

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.

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.

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.

4. Schwartz O. Cryopreservation as long-term storage of teeth for transplantation or replantation. Int J Oral Maxillofac Surg 1986. 15(1)30–32.

5. Pegg DE. The history and principles of cryopreservation. Semin Reprod Med 2002. 20(1)5–13.

6. Neulieb RL, Neulieb MK. The diverse actions of dimethyl sulphoxide: an indicator of membrane transport activity. Cytobios 1990. 63(254-255)139–165.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

24. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 1984. 247(3 Pt 1)C125–C142.

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.

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.

27. Rowley SD, Anderson GL. Effect of DMSO exposure without cryopreservation on hematopoietic progenitor cells. Bone Marrow Transplant 1993. 11(5)389–393.

28. Takahashi T, Hirsh A, Erbe E, Williams RJ. Mechanism of cryoprotection by extracellular polymeric solutes. Biophys J 1988. 54(3)509–518.

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.

30. Lakey JR, Rajotte RV, Fedorow CA, Taylor MJ. Islet cryopreservation using intracellular preservation solutions. Cell Transplant 2001. 10(7)583–589.

31. Chesne C, Guillouzo A. Cryopreservation of isolated rat hepatocytes: a critical evaluation of freezing and thawing conditions. Cryobiology 1988. 25(4)323–330.

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.

33. Meng Q. Hypothermic preservation of hepatocytes. Biotechnol Prog 2003. 19(4)1118–1127.

34. Shimono M, Lijima K. Pathology of Wound Healing <Clinical Aspect> Transplantation and Replantation of Teeth-Practical Use of Periodontal Ligament- 1995. Korean Editionth ed. 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;

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