J Korean Acad Conserv Dent.  2010 Jul;35(4):285-294. 10.5395/JKACD.2010.35.4.285.

The evaluation of periodontal ligament cells of rat teeth after low-temperature preservation under high pressure

• Chung JH
• Kim J
• Choi SH
• Kim ES
• Park J
• Lee SJ

• KMID: 2176391
• DOI: http://doi.org/10.5395/JKACD.2010.35.4.285

Abstract

The purpose of this study was to evaluate the viability of periodontal ligament cells of rat teeth after low-temperature preservation under high pressure by means of MTT assay, WST-1 assay. 12 teeth of Sprague-Dawley white female rats of 4 week-old were used for each group. Both side of the first and second maxillary molars were extracted as atraumatically as possible under tiletamine anesthesia. The experimental groups were group 1 (Immediate extraction), group 2 (Slow freezing under pressure of 3 MPa), group 3 (Slow freezing under pressure of 2 MPa), group 4 (Slow freezing under no additional pressure), group 5 (Rapid freezing in liquid nitrogen under pressure of 2 MPa), group 6 (Rapid freezing in liquid nitrogen under no additional pressure), group 7 (low-temperature preservation at 0degrees C under pressure of 2 MPa), group 8 (low-temperature preservation at 0degrees C under no additional pressure), group 9 (low-temperature preservation at -5degrees C under pressure of 90 MPa). F-medium and 10% DMSO were used as preservation medium and cryo-protectant. For cryo-preservation groups, thawing was performed in 37degrees C water bath, then MTT assay, WST-1 assay were processed. One way ANOVA and Tukey HSD 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 7 (0degrees C/2 MPa) showed higher viability of periodontal ligament cells than other group (2-6, 8) and this was statistically significant (p < 0.05), but showed lower viability than group 1, immediate extraction group (no statistical significance). By the results of this study, low-temperature preservation at 0degrees C under pressure of 2 MPa suggest the possibility for long term preservation of teeth.

Keyword

Periodontal ligament cell; MTT; WST-1; Viability; Low temperature preservation under pressure; Rapid freezing; Cryopreservation

MeSH Terms

Anesthesia
Animals
Baths
Cryopreservation
Dimethyl Sulfoxide
Eosine Yellowish-(YS)
Female
Freezing
Humans
Molar
Nitrogen
Periodontal Ligament
Rats
Tiletamine
Tooth
Dimethyl Sulfoxide
Eosine Yellowish-(YS)
Nitrogen
Tiletamine

Figure

• 

  Figure 1 Schematic diagram of program freezer with pressure vessel. a. Oxygen container: 2 or 3 MPa of pressure, b. Program freezer, c. Pressure bottle, d. 2 ml Cryotube: 1 ml F medium + 10% DMSO, e. Tooth treated 10% DMSO, f. Pressure valve, g. Thermometer.

Reference

1. Schwartz O, Andreasen JO. Cryopreservation of mature teeth before replantation in monkeys(I). Effect of different cryopreservation agents and freezing devices. Int J Oral Surg. 1983. 12:425–436.

2. 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. 14:350–361.

3. Kristerson L. Autotransplantation of human premolars. A clinical and radiographic study of 100 teeth. Int J Oral Surg. 1985. 14:200–213.

4. Lindskog S, Blomlof L, Hammarstrom L. Repair of periodontal tissues in vivo and in vitro. J Clin Periodontol. 1983. 10:188–205.

5. Melcher AH. Repair of wounds in the periodontium of the rat. Influence of periodontal ligament on osteogenesis. Arch Oral Biol. 1970. 15:1183–1204.

6. Andreasen JO. Interrelation between alveolar bone and periodontal ligament repair after replantation of mature permanent incisors in monkeys. J Periodont Res. 1981. 16:228–235.

7. Andreasen J.O0, 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.

8. Schwartz O, Rank C.P. "Autotransplantation of cryopreserved tooth in connection with orthodontic treatment". Am J Orthod Dentofacial Orthop. 1986. 90(1):67–72.

9. Schwartz O. Cryopreservation of teeth before replantation or transplantation. Atlas of replantation and transplantation of teeth. 1992. Medglobe;241–256.

10. Rubinsky B. Principles of low temperature cell preservation. Heart Fail Rev. 2003. 8(3):277–284.

11. Pegg DE. The history and principles of cryopreservation. Seminars in reproductive medicine. 2002. 20(1):5–13.

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

13. Kawasaki N, Hamamoto Y, Nakajima T, Irie K, Ozawa H. Periodontal regeneration of transplanted rat molars after cryopreservation. Archives of Oral Biology. 2004. 49:59–69.

14. Lee YE, Kim ES, Kim J, Han SH, Lee SJ. Efficacy of programmed cryo-preservation under pressure in rat periodontal ligament cells. J Korean Acad Conserv Dent. 2009. 34:356–363.

15. Schluter O, Urrutia Benet G, Heinz V, Knorr D. Metastable states of water and ice during pressure-supported freezing of potato tissue. Biotechnol Prog. 2004. 20:799–810.

16. Arav A, Natan Y. Directional freezing: a solution to the methodological challenges to preserve large organs. Semin Reprod Med. 2009. 11. 27(6):438–442. Epub Oct 5, 2009.

17. Inuzuka K, Unno N, Yamamoto N, Sagara D, Suzuki M, Nishiyama M, Konno H. Effect of hyperbarically oxygenated-perfluorochemical with university of Wisconsin solution on preservation of rat small intestine using an original pressure-resistant portable apparatus. Surgery. 2007. 142(1):57–66.

18. Takahashi T, Kakita A, Takahashi Y, Sakamoto I, Yokoyama K, Fujiu T, Yamashina S, Tamaki T, Takazawa Y, Muratsubaki R. Funtional integrity of the rat liver after subzero preservation under high pressure. Transplantation Proceedings. 2000. 32:1634–1636.

19. Takahashi T, Kakita A, Takahashi Y, Yokoyama K, Sakamoto I, Yamashina S. Preservation of rat livers by supercooling under high pressure. Transplantation Proceedings. 2001. 33:916–919.

20. Pribenszky C, Molnar M, Cseh S, Solti L. Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Anim Reprod Sci. 2005. 87(1-2):143–150.

21. Pribenszky C, Du Y, Molnar M, Harnos A, Vajta G. Increased stress tolerance of matured pig oocytes after high hydrostatic pressure treatment. Anim Reprod Sci. 2008. 106(1-2):200–207.

22. Du Y, Lin L, Schmidt M, Bogh IB, Kragh PM, Sorensen CB, Li J, Purup S, Pribenszky C, Molnar M, Kuwayama M, Zhang X, Yang H, Bolund L, Vajta G. High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival. Cloning and stem cells. 2008. 10(3):325–330.

23. Du Y, Pribenszky C, Molnar M, Zhang X, Yang H, Kuwayama M, Pedersen AM, Villemoes K, Bolund L, Vajta G. High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification. Reproduction. 2008. 135(1):13–17.

24. Fowler A. Cryo-injury and biopreservation. Ann N Y Acad Sci. 2005. Dec. 1066:119–135.

25. Gao D, Critser JK. Mechanisms of cryoinjury in living cells. ILAR J. 2000. 41(4):187–196.

26. 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.

27. Hyon SH. A Non-frozen living tissue bank for allotransplantation using green tea polyphenols. Yonsei Med J. 2004. 45(6):1025–1034.

28. Bridgman P.W. Water in the liquid and five solid forms under pressure. Proc Amer Acad Arts and Sci. 1911. 47:441–558.

29. Pribenszky C, Molnár M, Cseh S, Solti L. Survival of mouse blastocysts after low-temperature preservation under high pressure. Acta Vet Hung. 2004. 52(4):479–487.

30. Baek DY, Lee SJ, Jung HS, Kim ES. Comparison of viability of oral epithelial cells stored by different freezing methods. J Korean Acad Conserv Dent. 2009. 34:491–499.

31. Andreasen JO, Schwartz O. Atlas of replantation and transplantation of teeth. 1992. Fribourg, Switzerland: Mediglobe SA.

32. Kawata T. Tooth transplantation by teeth bank-approach to human-Hiroshima. 2005. Department of Orthodontics, Hiroshima University School of Dentistry.

33. Kaku M, Kamata H, Kawata T. Cryopreservation of PDL cells by use of program freezer with magnetic field for tooth banking. Dentistry in Japan. 2007. 43:82–86.

34. Ashwood-smith MJ. Low temperature preservation of cells, tissues and organs. Pitman Medicals. 1980. 19–45.

35. Farrant J. Water transport and cell survival in cryobiological procedures. Philos Trans R Soc Lond B Biol Sci. 1977. 278(959):191–205.

36. Kim JW, Kim ES, Kim J, Lee SJ. Evaluation of periodontal ligament cell viability in rat teeth after frozen preservation using in-vivo MTT assay. J Korean Acad Conserv Dent. 2006. 3(3):192–202.

37. Abe F, Kato C, Horikoshi K. Pressure regulated metabolism in microorganisms. Trends Microbiol. 1999. 7:447–453.

38. Aldridge BE, Bruner LJ. Pressure effects on mechanisms of charge transport across bilayer membranes. Biochem Biophys Acta. 1985. 817:343–354.

39. Huang SY, Pribenszky C, Kuo YH, et al. The effect of hydrostatic pressure treatment on the protein profile of boar spermatozoa before and after freezing. Proceedings of the 6th International Conference on Boar Semen Preservation. 2007. Alliston, Ontario, Cananda. I.-34.

40. Kaarniranta K, Elo M, Sironen R, Lammi MJ, Goldring MB, Eriksson JE, Sistonen L, Helminen HJ. Hsp 70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation. Proc Natl Acad Sci USA. 1998. 95:2319–2324.

41. Elo MA, Sironen RK, Karjalainen HM, Kaarniranta KK, Helminen HJ, Lammi MJ. Specific induction of heat shock protein 90 beta by high hydrostatic pressure. Biorheology. 2003. 40:141–146.

42. Alahiotis SN. Heat shock proteins. A new view on the temperature compensation. Comp Biochem Physiol B. 1983. 75:379–387.

43. Parsell DA, Lindquist S. The function of heat shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Ann Rev Genet. 1993. 27:437–496.

44. Kwak HJ, Jun CD, Pae HO, Yoo JC, Park YC, Choi BM, Na YG, Park RK, Chung HT, Park WJ, Seo JS. The role of inducible 70-kDa heat shock protein in cell cycle control, differentiation and apoptotic cell death of the human myeloid leukemic HL-60 cells. Cell Immunol. 1998. 187:1–12.

45. Carlson MA. Technical note: assay of cell quantity in the fibroblast populated collagen matrix with a tetrazolium reagent. Eur Cell Mater. 2006. 12:44–48.

46. Kim ES, Jeon IS, Kim JW, Kim J, Juhn HS, Lee SJ. An MTT-based method for quantification of periodontal ligament cell viability. Oral Diseases. 2007. 13(5):495–499.

47. Ahn HJ, Kim ES, Kim J, Kim DW, Kim KY, Lee CY, Lee SJ. Evaluation of viability of periodontal ligament cell in rat teeth using slow cryoperservation method with magnetic field. J Korean Acad Conserv Dent. 2008. 33(4):332–340.

48. Alotto D, Ariotti S, Graziano S, Verrua R, Stella M, Magliacani G, Castagnoli C. The role of quality control in a skin bank: tissue viability determination. Cell and Tissue banking. 2002. 3:3–10.