A HISTOLOGIC STUDY OF BONE FORMATION ACCORDING TO DIFFERENT CRYOPROTECTANTS DURING CRYOPRESERVATION OF BONE

동결보호제의 종류에 따른 냉동보관자가골의 골형성능에 대한 연구

  • Park, Hyun-Wook (Dept. of Oral and Maxillofacial Surgery, Division of Dentistry Graduate School, Kyung-Hee University) ;
  • Lee, Baek-Soo (Dept. of Oral and Maxillofacial Surgery, Division of Dentistry Graduate School, Kyung-Hee University)
  • 박현욱 (경희대학교 대학원 치의학과 구강악안면외과학교실) ;
  • 이백수 (경희대학교 대학원 치의학과 구강악안면외과학교실)
  • Published : 2005.06.30

Abstract

Purpose: Several cryoprotectants are in use to help the survival of cells during cryopreservation of bone in maxillofacial region. Among them, $Me_2SO$(dimethyl sulfoxide), EG(ethylene glycol), sucrose were used for experimentally created defects with accompanying cryopreserved bone graft in the rabbit model. The aim of this study is to analyze the effect of above mentioned agents on bone formation using histologic and histomorphometrical methods, thus to provide experimental support for clinical application of these agents. Materials and methods: Nine rabbits were used as experimental animals. Surgical defects were created on the distal femoral heads and mesial tibial heads of each animal using trephine drill(5mm diameter and 5mm length). The harvested bones were cryopreserved in $-80^{\circ}C$ refrigerator for one week. The defects were filled with cryopreserved bone with cryoprotectants as experimental groups and cryopreserved bone without cryoprotectant as control. Then, the animals were sacrificed at 1, 2, and 3 weeks after surgery. With Goldner's modified Masson trichrome staining and semiautomatic image analysis system, we observed the change of the cells and bone formation. Results: After bone graft, bone formation and active remodeling process were examined in all experimental groups and the control. But the intensity of such activities of the control were somewhat weaker than that of the experiments. Especially $Me_2SO$+sucrose group was the best in bone formation and bone remodeling. $Me_2SO$ group was more than that of EG group in bone fomation. Sucrose seems to be helpful in survival of the bone cell. Histologic findings showed superior bony quantity and quality in experimental groups than that in control. Conclusions: The data from this study provides the basis for future studies for evaluating the effect of cryoprotectants in the cryopreservation of bone and clinical study for predictable use of these agents.

Keywords

References

  1. Bakken AM, Bruserud O, Abrahamsen JF: No Differences in Colony formation of Peripheral blood stem cells Frozen with 5% or 10% Dimethyl Sulfoxide. J Hematother Stem Cell Res 2003;12(3):341 https://doi.org/10.1089/152581603322023070
  2. Jomha NM, Lavoie G: Cryopreservation of Intact Human Articular Cartilage. Acta Crystallogr D Biol Crystllogr 2002;58:2060 https://doi.org/10.1107/S0907444902017183
  3. Lee JA: Rapid Freezing of Umbilical cord Blood in a -$80^{\circ}C$. The Korean J Blood trasfusion 2001;1(1):55
  4. Jung YH: Cryopreservtion of Rat Hepatocytes for the Use of Primary Culture. The Korean J Gastroenterology 2003;41(1):41
  5. Erdmann L, Ehlers N: Long-term Results with Organ Cultered, Cryopreserved Human Corneal Grafts. Re-examination of 17 Patients. Acta Opthal 1993;71:703
  6. Amorim CA, Rondina D, Rodrigues AP, Costa SH, Goncalves PB, de Figueiredo JR, Giorgetti A: Isolated Ovine Primordial Follicles Cryopreserved in Different Concentrations of Ethyelene Glycol. Theriogenology 2003;60:735 https://doi.org/10.1016/S0093-691X(03)00089-X
  7. Mantovani R, Rora A, Falomo ME, Bailoni L, Vincenti L: Comparison Between Glycerol and Etylene Glycol for the Cryopreservation of Equine Spermatozoa. Reprod Nutr 2002 ;42:217 https://doi.org/10.1051/rnd:2002020
  8. Buhr MM, Fiser P, Bailey JL, Curtis EF: Cryopreservation in Different concentrations of Glycerol alters Boar sperm and Their membranes. hematother Stem Cell Res 2001;10(5):709 https://doi.org/10.1089/152581601753193931
  9. Camus M, Abbeel E, Waesberghe L, Wisanto A, Devroey P, Steirteghem AC: Human Embryo Viability after Freezing with Dimethylsulfoxide as a ryoprotectant. Fertil Steril 1989;51(3):460
  10. Browne RK, Mahony M, Clulow J: A Aomparison of Sucrose, Saline, and Saline with egg-yolk diluents on the Cryopreservation of Cane toad Sperm. Exp Anim 2002;51(4):383
  11. Pegg DE: The History and Principles of Cryopreservation. J Androl 2001;22(6):961
  12. Brondet R, Gilbert-thevenin A, Pierre C, Ehrsam A: Skin Preservation by Programmed Freezing. Br J Plast Surg 1982;35:530
  13. Ishine N, Rubonsky B, Lee CY: A Histological Analysis of Liver Injury in Freezing Storage. Cryobiology 1999;39(3):217
  14. Negishi T, Ishii Y: Cryopreservation of Brain Tissue for Primary Culture. Hum Reprod 2002;17(9):2419 https://doi.org/10.1093/humrep/17.9.2419
  15. Tomford WW, Ploetz JE, Mankin HJ: Bone Allografts of Femoral Heads ; Procurement and Storage. J Bone Joint Surg 1986;68A(4):534
  16. Mazur P, Leibo SP, Chu EHY: A Two-factor Hypothesis of Freezing Injury. Exp Cell Res 1972;71:345
  17. Lovelock JE: The Mechanism of the Protective Action of Glycerol Against Haemolysis by freezing and Thawing. Biochem Biophys Acta 1953;11:28
  18. Lovelock JE, Bishop M: Prevention of Freezing Damage to Living Cells by Dimethyl Sulfoxide. Nature 1959;183:1394 https://doi.org/10.1038/1831394a0
  19. Ashwood-Smith MJ: Current Concepts Concerning Radioprotective and Cryoprotective Properties of Diemthyl Sulfoxide in Cellular Systems. Ann N.Y. Acak Sci 1967;141:41
  20. Ashwood-Smith A: Preservation of Mouse Bone Marrow at -78 with Dimethyl Sulfoxide. Nature 1961;190:1204 https://doi.org/10.1038/1901204a0
  21. Dougherty R: Use of Dimethyl Sulfoxide for Preservation of Tissue Culture Cells by Freezing. Nature 1962;193:550 https://doi.org/10.1038/193550a0
  22. 장영진: Pinctada fucata martensil 담륜자의 냉동보존을 위한 동해 방지제의 선택. 발생과 생식 1991;3:107
  23. Salehnia M: Autograft of Vitrified Mouse Ovaries using Ethylene Glycol as Cryoprotectant. Exp Anim 2002;51 :509 https://doi.org/10.1538/expanim.51.509
  24. Robinson MP, Wusteman MC, Wang L, Pegg DE: Electromagnetic Re-warming of Cryopreserved Tissue: Effect of Choice of Cryoprotectant and Sample Shape on Uniformity of Heating. Semin Reprod Med 2002;20(1):1
  25. Jung-Hwan Oh, Joachim E. Zller and Alexander Kbler: A new bone banking technique to maintain osteoblast viability in frozen human iliac cancellous bone. Cryobiology 2002;44:279
  26. Leibo SP: A One-Step Method for Direct Non-surgical Transfer of Frozen-Thawed Bovine Embryos. Theriogenology 1984;21:767
  27. Schneider U, Mazur P: Osmotic Consequences of Cryoprotectant Perneability and its Relation to Survival of Frozen-Thawed Embryos. Theriogenology 1984;21:68
  28. Boyne PJ: Review of the Literature on Cryopreservation of Bone. Cryobiology 1968;4:341 https://doi.org/10.1016/S0011-2240(68)80134-8
  29. Willadson SM: Factors affecting the Survival of Sheep Embryos during Deep-Freezing and Thawing. In the Freezing of Mammalian Embryos. Edited by Elliot K, Whelan J, Excepta Medica/Amsterdam, 1977
  30. Rall WF, Fathy GM: Ice Free Cryopreservation of Mouse Embryos by Vitrification. Nature 1985;313:573 https://doi.org/10.1038/313573a0
  31. Ohlendorf C, Tomford WW, Markin HJ: Chondrocyte Survival in Cryopreserved Osteochondral Articular Cartilage. J Orthop Res 1996;14(3): 413 https://doi.org/10.1002/jor.1100140311
  32. Goldberg VM, Shaffer JW, Field G, Davy DT: Biology of Vascularized Bone Grafts. Orthoped Clin North Am 1987;18(2):197
  33. Burwell RG: The Function of Bone Marrow in the Incorporation of a Bone Graft. Clin Orthopae Rel Res 1985;200:125
  34. Frielander GE: Immune Responses to Osteochondral Allografts: Current Knowledge and Future Directions. Clin Orthop 174:58, 1983
  35. Elves MW: Humoral Immune Response og Allografts of bone. Int Arch Allergy Appl Immunol 1974;47:718
  36. Darcy DA: Reaction of Rabbits to Frozen Homografts. Pathol Bacteriol 1955;71:143
  37. Sell KW, Friedlaender GE, Strong DM: Immunogenicity and Freezedrying. INSERM 1976;62:187
  38. Solheim E, Pinholt EM, Talsnes O et al: The Relationship between Revascularization and Osteogenesis in Fresh or Demineralized Bone Grafts. Eur Surg Res 2001;33:42 https://doi.org/10.1159/000049728
  39. Bassett CAL: Clinical Implications of Cell Function in Bone Grafting. Clin Orthop Rel Res 1972;87:49
  40. Epstein S: Serum and Urinary Markers of Bone Remodelling: Assessment of Bone Turnover. Endocrine Rev 1988;9:437 https://doi.org/10.1210/edrv-9-4-437
  41. Harris SA, Enger RJ, Riggs BL, Spelsberg TC: Development and Charicterization of a Conditionally Immortalized Human Fetal Osteoblastic Cell Line. J Bone Miner Res 1995;10(2):178 https://doi.org/10.1002/jbmr.5650100203
  42. Auxhausen W: The Osteogenetic Phases of Regenerations of Bone, a Historical and Experimental study. J Bone Joint Surg 1956;38A:593
  43. Trounson A: Cryopreservation. Br Med Bulletin 1990;46(3):695
  44. Whittingham DG, Leibo SP, Mazur P: Survival of Mouse Embryos Frozen to -196oC and -296oC. Science 1972;178:411 https://doi.org/10.1126/science.178.4059.411
  45. Szell A, Shelton JN: Sucrose Dilution of Glycerol from Mouse Embryos Frozen Rapidly in Liquid Nitrogen Vapour. J Reprod Fert 1986;76:401 https://doi.org/10.1530/jrf.0.0760401