Cell and Tissue Banking

, Volume 14, Issue 2, pp 231–242 | Cite as

The effect of sterilization on the mechanical properties of intact rabbit humeri in three-point bending, four-point bending and torsion

  • Nicholas A. Russell
  • Alain Rives
  • Matthew H. Pelletier
  • Warwick J. Bruce
  • William R. WalshEmail author
Original Paper


Load bearing bone allografts are used to replace the mechanical function of bone that has been removed or to augment bone that has been damaged in trauma. In order to minimize the risk of infection and immune response, the bone is delipidated and terminally sterilized prior to implantation. The optimal method for bone graft sterilization has been the topic of considerable research. Recently, supercritical carbon dioxide (SCCO2) treatments have been shown to terminally sterilize bone against a range of bacteria and viruses. This study aimed to evaluate the effect of SCCO2 treatment compared with two doses of gamma irradiation, on the mechanical properties of whole bone. Paired rabbit humeri were dissected and randomly assigned into either SCCO2 control, SCCO2 additive or gamma irradiation at 10 or 25 kGy treatment groups. The bones were mechanically tested in three-point and four-point bending and torsion, with the lefts acting as controls for the treated rights. Maximum load, energy to failure and stiffness were evaluated. This study found that SCCO2 treatment with or without additive did not alter maximum load, energy to failure or stiffness significantly under any loading modality. Gamma irradiation had a deleterious dose dependant effect, with statistically significant decreases in all mechanical tests at 25 kGy; while at 10 kGy there were reductions in all loading profiles, though only reaching statistical significance in torsion. This study highlights the expediency of SCCO2 treatment for bone allograft processing as terminal sterilization can be achieved while maintaining the intrinsic mechanical properties of the graft.


Allograft Sterilization Supercritical fluid Gamma irradiation Mechanical 


Conflict of interest

The authors declare that there is no conflict of interest in the preparation of this manuscript.


  1. Akkus O, Belaney RM (2005) Sterilization by gamma radiation impairs the tensile fatigue life of cortical bone by two orders of magnitude. J Orthop Res 23(5):1054–1058. doi: 10.1016/j.orthres.2005.03.003 PubMedCrossRefGoogle Scholar
  2. Akkus O, Rimnac CM (2001) Fracture resistance of gamma radiation sterilized cortical bone allografts. J Orthop Res 19(5):927–934. doi: 10.1016/S0736-0266(01)00004-3 PubMedCrossRefGoogle Scholar
  3. Akkus O, Belaney RM, Das P (2005) Free radical scavenging alleviates the biomechanical impairment of gamma radiation sterilized bone tissue. J Orthop Res 23(4):838–845. doi: 10.1016/j.orthres.2005.01.007 PubMedCrossRefGoogle Scholar
  4. Anderson MJ, Keyak JH, Skinner HB (1992) Compressive mechanical properties of human cancellous bone after gamma irradiation. J Bone Joint Surg Am 74(5):747–752PubMedGoogle Scholar
  5. Balsly C, Cotter A, Williams L, Gaskins B, Moore M, Wolfinbarger L (2008) Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts. Cell Tissue Bank 9(4):289–298PubMedCrossRefGoogle Scholar
  6. Barry JJ, Silva MM, Popov VK, Shakesheff KM, Howdle SM (2006) Supercritical carbon dioxide: putting the fizz into biomaterials. Philos Transact A Math Phys Eng Sci 364(1838):249–261. doi: 10.1098/rsta.2005.1687 PubMedCrossRefGoogle Scholar
  7. Bertoloni G, Bertucco A, De Cian V, Parton T (2006) A study on the inactivation of micro-organisms and enzymes by high pressure CO2. Biotechnol Bioeng 95(1):155–160. doi: 10.1002/bit.21006 PubMedCrossRefGoogle Scholar
  8. Burstein AH, Zika JM, Heiple KG, Klein L (1975) Contribution of collagen and mineral to the elastic-plastic properties of bone. J Bone Joint Surg Am 57(7):956–961PubMedGoogle Scholar
  9. Campbell DG, Li P, Stephenson AJ, Oakeshott RD (1994) Sterilization of HIV by gamma irradiation. A bone allograft model. Int Orthop 18(3):172–176PubMedCrossRefGoogle Scholar
  10. Cornu O, Banse X, Docquier PL, Luyckx S, Delloye C (2000) Effect of freeze-drying and gamma irradiation on the mechanical properties of human cancellous bone. J Orthopaed Res 18(3):426–431. doi: 10.1002/jor.1100180314 CrossRefGoogle Scholar
  11. Cornu O, Boquet J, Nonclercq O, Docquier PL, Van Tomme J, Delloye C, Banse X (2011) Synergetic effect of freeze-drying and gamma irradiation on the mechanical properties of human cancellous bone. Cell Tissue Bank 12(4):281–288. doi: 10.1007/s10561-010-9209-1 PubMedCrossRefGoogle Scholar
  12. Currey JD, Foreman J, Laketic I, Mitchell J, Pegg DE, Reilly GC (1997) Effects of ionizing radiation on the mechanical properties of human bone. J Orthopaed Res 15(1):111–117. doi: 10.1002/jor.1100150116 CrossRefGoogle Scholar
  13. Davies OR, Lewis AL, Whitaker MJ, Tai H, Shakesheff KM, Howdle SM (2008) Applications of supercritical CO2 in the fabrication of polymer systems for drug delivery and tissue engineering. Adv Drug Deliv Rev 60(3):373–387. doi: 10.1016/j.addr.2006.12.001 PubMedCrossRefGoogle Scholar
  14. Davy DT (1999) Biomechanical issues in bone transplantation. Orthop Clin North Am 30(4):553–563PubMedCrossRefGoogle Scholar
  15. DePaula CA, Truncale KG, Gertzman AA, Sunwoo MH, Dunn MG (2005) Effects of hydrogen peroxide cleaning procedures on bone graft osteoinductivity and mechanical properties. Cell Tissue Bank 6(4):287–298. doi: 10.1007/s10561-005-3148-2 PubMedCrossRefGoogle Scholar
  16. Dillow AK, Dehghani F, Hrkach JS, Foster NR, Langer R (1999) Bacterial inactivation by using near- and supercritical carbon dioxide. Proc Natl Acad Sci USA 96(18):10344–10348PubMedCrossRefGoogle Scholar
  17. Dziedzic-Goclawska A, Ostrowski K, Stachowicz W, Michalik J, Grzesik W (1991) Effect of radiation sterilization on the osteoinductive properties and the rate of remodeling of bone implants preserved by lyophilization and deep-freezing. Clin Orthop Relat Res 272:30–37PubMedGoogle Scholar
  18. Dziedzic-Goclawska A, Kaminski A, Uhrynowska-Tyszkiewicz I, Stachowicz W (2005) Irradiation as a safety procedure in tissue banking. Cell Tissue Bank 6(3):201–219. doi: 10.1007/s10561-005-0338-x PubMedCrossRefGoogle Scholar
  19. Fages J, Marty A, Delga C, Condoret JS, Combes D, Frayssinet P (1994) Use of supercritical CO2 for bone delipidation. Biomaterials 15(9):650–656PubMedCrossRefGoogle Scholar
  20. Fages J, Poirier B, Barbier Y, Frayssinet P, Joffret ML, Majewski W, Bonel G, Larzul D (1998) Viral inactivation of human bone tissue using supercritical fluid extraction. ASAIO J 44(4):289–293PubMedCrossRefGoogle Scholar
  21. Fideler BM, Vangsness CT Jr, Lu B, Orlando C, Moore T (1995) Gamma irradiation: effects on biomechanical properties of human bone-patellar tendon-bone allografts. Am J Sports Med 23(5):643–646PubMedCrossRefGoogle Scholar
  22. Freeman JJ, Silva MJ (2002) Separation of the Raman spectral signatures of bioapatite and collagen in compact mouse bone bleached with hydrogen peroxide. Appl Spectrosc 56(6):770–775CrossRefGoogle Scholar
  23. Gerd B (2005) Supercritical fluids: technology and application to food processing. J Food Eng 67(1–2):21–33. doi: 10.1016/j.jfoodeng.2004.05.060 Google Scholar
  24. Gibbons MJ, Butler DL, Grood ES, Bylski-Austrow DI, Levy MS, Noyes FR (1991) Effects of gamma irradiation on the initial mechanical and material properties of goat bone-patellar tendon-bone allografts. J Orthop Res 9(2):209–218. doi: 10.1002/jor.1100090209 PubMedCrossRefGoogle Scholar
  25. Godette GA, Kopta JA, Egle DM (1996) Biomechanical effects of gamma irradiation on fresh frozen allografts in vivo. Orthopedics 19(8):649–653PubMedGoogle Scholar
  26. Grieb TA, Forng R-Y, Stafford RE, Lin J, Almeida J, Bogdansky S, Ronholdt C, Drohan WN, Burgess WH (2005) Effective use of optimized, high-dose (50 kGy) gamma irradiation for pathogen inactivation of human bone allografts. Biomaterials 26(14):2033–2042PubMedCrossRefGoogle Scholar
  27. Hamer AJ, Strachan JR, Black MM, Ibbotson CJ, Stockley I, Elson RA (1996) Biochemical properties of cortical allograft bone using a new method of bone strength measurement. A comparison of fresh, fresh-frozen and irradiated bone. J Bone Joint Surg Am 78(3):363–368Google Scholar
  28. Hamer AJ, Stockley I, Elson RA (1999) Changes in allograft bone irradiated at different temperatures. J Bone Joint Surg Am 81(2):342–344CrossRefGoogle Scholar
  29. Hemmer J (2007) Sterilization of bacterial spores by using supercritical carbon dioxide and hydrogen peroxide. J Biomed Mater Res A 80B:511–518CrossRefGoogle Scholar
  30. Hubner W, Blume A, Pushnjakova R, Dekhtyar Y, Hein HJ (2005) The influence of X-ray radiation on the mineral/organic matrix interaction of bone tissue: an FT-IR microscopic investigation. Int J Artif Organs 28(1):66–73PubMedGoogle Scholar
  31. Ijiri S, Yamamuro T, Nakamura T, Kotani S, Notoya K (1994) Effect of sterilization on bone morphogenetic protein. J Orthop Res 12(5):628–636. doi: 10.1002/jor.1100120505 PubMedCrossRefGoogle Scholar
  32. Jinno T, Miric A, Feighan J, Kirk SK, Davy DT, Stevenson S (2000) The effects of processing and low dose irradiation on cortical bone grafts. Clin Orthop Relat Res 375:275–285PubMedCrossRefGoogle Scholar
  33. Lewandrowski KU, Gresser JD, Bondre S, Silva AE, Wise DL, Trantolo DJ (2000) Developing porosity of poly(propylene glycol-co-fumaric acid) bone graft substitutes and the effect on osteointegration: a preliminary histology study in rats. J Biomater Sci Polym Ed 11(8):879–889PubMedCrossRefGoogle Scholar
  34. Mikhael MM, Huddleston PM, Zobitz ME, Chen Q, Zhao KD, An K-N (2008) Mechanical strength of bone allografts subjected to chemical sterilization and other terminal processing methods. J Biomech 41(13):2816–2820. doi: 10.1016/j.jbiomech.2008.07.012 PubMedCrossRefGoogle Scholar
  35. Mitchell EJ, Stawarz AM, Kayacan R, Rimnac CM (2004) The effect of gamma radiation sterilization on the fatigue crack propagation resistance of human cortical bone. J Bone Joint Surg Am 86-A(12):2648–2657PubMedGoogle Scholar
  36. Mitton D, Rappeneau J, Bardonnet R (2005) Effect of a supercritical CO2 based treatment on mechanical properties of human cancellous bone. Eur J Orthop Surg Traumatol 15(4):264–269. doi: 10.1007/s00590-005-0250-x CrossRefGoogle Scholar
  37. Nguyen H, Morgan DA, Forwood MR (2007a) Sterilization of allograft bone: effects of gamma irradiation on allograft biology and biomechanics. Cell Tissue Bank 8(2):93–105. doi: 10.1007/s10561-006-9020-1 PubMedCrossRefGoogle Scholar
  38. Nguyen H, Morgan DA, Forwood MR (2007b) Sterilization of allograft bone: is 25 kGy the gold standard for gamma irradiation? Cell Tissue Bank 8(2):81–91. doi: 10.1007/s10561-006-9019-7 PubMedCrossRefGoogle Scholar
  39. Nichols A, Burns D, Christopher R (2009) The sterilization of human bone and tendon musculoskeletal allograft tissue using supercritical carbon dioxide. J Orthopaed 6(2):9–17Google Scholar
  40. Qiu Q-Q, Leamy P, Brittingham J, Pomerleau J, Kabaria N, Connor J (2009) Inactivation of bacterial spores and viruses in biological material using supercritical carbon dioxide with sterilant. J Biomed Mater Res B 91B(2):572–578. doi: 10.1002/jbm.b.31431 CrossRefGoogle Scholar
  41. Salehpour A, Butler DL, Proch FS, Schwartz HE, Feder SM, Doxey CM, Ratcliffe A (1995) Dose-dependent response of gamma irradiation on mechanical properties and related biochemical composition of goat bone-patellar tendon-bone allografts. J Orthop Res 13(6):898–906. doi: 10.1002/jor.1100130614 PubMedCrossRefGoogle Scholar
  42. Schwiedrzik JJ, Kaudela KH, Burner U, Zysset PK (2011) Fabric-mechanical property relationships of trabecular bone allografts are altered by supercritical CO2 treatment and gamma sterilization. Bone 48(6):1370–1377. doi: 10.1016/j.bone.2011.03.768 PubMedCrossRefGoogle Scholar
  43. Shieh E, Paszczynski A, Wai CM, Lang Q, Crawford RL (2009) Sterilization of Bacillus pumilus spores using supercritical fluid carbon dioxide containing various modifier solutions. J Microbiol Meth 76(3):247–252CrossRefGoogle Scholar
  44. Simonian PT, Conrad EU, Chapman JR, Harrington RM, Chansky HA (1994) Effect of sterilization and storage treatments on screw pullout strength in human allograft bone. Clin Orthop Relat Res 302:290–296PubMedGoogle Scholar
  45. Spilimbergo S, Bertucco A (2003) Non-thermal bacterial inactivation with dense CO(2). Biotechnol Bioeng 84(6):627–638. doi: 10.1002/bit.10783 PubMedCrossRefGoogle Scholar
  46. Spilimbergo S, Dehghani F, Bertucco A, Foster NR (2003) Inactivation of bacteria and spores by pulse electric field and high pressure CO2 at low temperature. Biotechnol Bioeng 82(1):118–125. doi: 10.1002/bit.10554 PubMedCrossRefGoogle Scholar
  47. Thoren K, Aspenberg P (1995) Ethylene oxide sterilization impairs allograft incorporation in a conduction chamber. Clin Orthop Relat Res 318:259–264PubMedGoogle Scholar
  48. Triantafyllou N, Sotiropoulos E, Triantafyllou JN (1975) The mechanical properties of the lyophylized and irradiated bone grafts. Acta Orthop Belg 41(suppl 1):35–44PubMedGoogle Scholar
  49. Vashishth D, Gibson GJ, Khoury JI, Schaffler MB, Kimura J, Fyhrie DP (2001) Influence of nonenzymatic glycation on biomechanical properties of cortical bone. Bone 28(2):195–201. doi: 10.1016/S8756-3282(00)00434-8 PubMedCrossRefGoogle Scholar
  50. Vastel L, Meunier A, Siney H, Sedel L, Courpied JP (2004) Effect of different sterilization processing methods on the mechanical properties of human cancellous bone allografts. Biomaterials 25(11):2105–2110. doi: 10.1016/j.biomaterials.2003.08.067 PubMedCrossRefGoogle Scholar
  51. Voggenreiter G, Ascherl R, Blumel G, Schmit-Neuerburg KP (1996) Extracorporeal irradiation and incorporation of bone grafts. Autogeneic cortical grafts studied in rats. Acta Orthop Scand 67(6):583–588PubMedCrossRefGoogle Scholar
  52. Watanabe T, Furukawa S, Hirata J, Koyama T, Ogihara H, Yamasaki M (2003) Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment. Appl Environ Microbiol 69(12):7124–7129PubMedCrossRefGoogle Scholar
  53. White A, Burns D, Christensen TW (2006) Effective terminal sterilization using supercritical carbon dioxide. J Biotechnol 123(4):504–515PubMedCrossRefGoogle Scholar
  54. Zhang J, Burrows S, Gleason C, Matthews MA, Drews MJ, Laberge M, An YH (2006) Sterilizing Bacillus pumilus spores using supercritical carbon dioxide. J Microbiol Methods 66(3):479–485. doi: 10.1016/j.mimet.2006.01.012 PubMedCrossRefGoogle Scholar
  55. Zhou Z, Qin T, Yang J, Shen B, Kang P, Peil F (2011) Mechanical strength of cortical allografts with gamma radiation versus ethylene oxide sterilization. Acta Orthop Belg 77(5):670–675PubMedGoogle Scholar
  56. Zioupos P, Currey JD, Hamer AJ (1999) The role of collagen in the declining mechanical properties of aging human cortical bone. J Biomed Mater Res 45(2):108–116. doi: 10.1002/(SICI)1097-4636(199905)45:2<108:AID-JBM5>3.0.CO;2-A PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Nicholas A. Russell
    • 1
  • Alain Rives
    • 1
  • Matthew H. Pelletier
    • 1
  • Warwick J. Bruce
    • 2
  • William R. Walsh
    • 1
    Email author
  1. 1.Surgical and Orthopaedic Research Laboratories, Prince of Wales Clinical School, Prince of Wales HospitalUniversity of New South WalesSydneyAustralia
  2. 2.Concord Repatriation General HospitalSydneyAustralia

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