Cell and Tissue Banking

, Volume 6, Issue 1, pp 25–31 | Cite as

Detection of living cells in non-processed but deep-frozen bone allografts

  • Ide  C. Heyligers
  • Jenneke Klein-Nulend


Impacted morselized donor bone is successfully used to treat bone loss in revision total hip arthroplasties. It is generally thought, but not proven, that the processing and storage at −80 °C of the donor bone kills all cells. Because of the risk of contamination and to increase our understanding about the process of new bone formation after revision total hip arthroplasty, the aim of this study was to investigate whether the donor bone does contain vital cells. Samples from 11 femoral heads were obtained according to the American and European standards of bone banking, and tested for their capacity to give rise to proliferating cells, using tissue culture methods. All bone samples were stored at  − 80°C for a minimum of 6 months. Bone sample cores were morselized and cultured for 6 weeks. Inverted phase contrast microscopy was used to evaluate cell growth. DNA marker analysis was used to confirm celluar identity.

All bank bone samples gave rise to cell growth. The cell cultures showed osteoblastic characteristics in that they expressed high levels of alkaline phosphatase activity. DNA marker analysis showed identical alleles for cultured cells from frozen bone and freshly obtained buccal cells from the same donor, indicating that the cells growing from the banked bone were indeed originating from the donor tissue. It was therefore concluded that −80 °C freezing of bone tissue does not routinely kill cells within the tissue.


Bone bank Bone cell Femoral head allograft Impaction grafting Total hip arthroplasty 


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  1. American Association of Tissue Banks 1996 Standards for Tissue Banking [editorial] American Association of Tissue BanksGoogle Scholar
  2. Aspenberg, P., Tagil, M., Kristensson, C., Lidin, S. 1996Bone graft proteins influence osteoconduction. A titanium chamber study in ratsActa Orthop. Scand.67377382PubMedGoogle Scholar
  3. Aubin, J.E., Liu, F. 1996The osteoblast lineageBilezikian, J.P.Raisz, L.G.Rodan, G.A. eds. Principles of Bone BiologyAcademic PressSan DiegoCA5168Google Scholar
  4. Beresford, J.N., Gallagher, J.A., Gowen, M., McGuire, M.K.B., Poser, J., Russell, R.G.G. 1983Human bone cells in culture. A novel system for the investigation of bone cell metabolismClin. Sci. (Colch)643839Google Scholar
  5. Biezen van, F.C., ten Have, B.L., Verhaar, J.A. 2000Impaction bone-grafting of severely defective femora in revision total hip surgery: 21 hips followed for 41–85 monthsActa Orthop. Scand.71135142Google Scholar
  6. Buma, P., Lamerigts, N., Schreurs, B.W., Gardeniers, J., Versleyen, D., Slooff, T.J. 1996Impacted graft incorporation after cemented acetabular revision Histological evaluation in 8 patientsActa Orthop. Scand.67536540PubMedCrossRefGoogle Scholar
  7. Burwell, R.G., Gowland, G. 1962aStudies in the transplantation of Bone III. The immune responses of lymph nodes draining components of fresh homologous cancellous bone and homologous bone treated by different methodsJ. Bone Joint Surg.44-B131Google Scholar
  8. Burwell, R.G. 1962bStudies in the transplantation of bone IV. The immune response of lymph nodes draining second-set homografts of fresh cancellous boneJ. Bone Joint Surg.44-B688Google Scholar
  9. Centers for Disease Control.1988Transmission of HIV through bone transplantation: case report and public health recommendationsMMWR Morb. Mortal. Wkly. Rep.37597599Google Scholar
  10. Centers for Disease Control.2003Hepatitis C virus transmissions from an antibody-negative organ and tissue donor – United States, 2002–2002MMWR Morb. Mortal. Wkly. Rep.52273276Google Scholar
  11. Conrad, E.U., Gretch, D.R., Obermeyer, K.R.,  et al. 1995Transmission of the hepatitis-C virus by tissue transplantationJ. Bone Joint Surg. Am.77214224PubMedGoogle Scholar
  12. Cook, S.D., Salkeld, S.L., Prewett, A.B. 1995Simian immunodeficiency virus (human HIV-II) transmission in allograft bone proceduresSpine2013381342CrossRefPubMedGoogle Scholar
  13. European Association for Musculo Skeletal Transplantation. 1997 Common Standards for Musculoskeletal Tissue Banking [editorial] European Association for Musculo Skeletal TransplantationGoogle Scholar
  14. Friedlaender, G.E. 1991Bone allografts: the biological consequences of immunological events [editorial]J. Bone Joint Surg. Am.7311191122PubMedGoogle Scholar
  15. Goldberg, V.M., Stevenson, S. 1987Natural history of autografts and allograftsClin. Orthop.716Google Scholar
  16. Jeffreys, A.J., Wilson, V., Thein, S.L. 1985Hypervariable ‘minisatellite’ regions in human DNANature3146773PubMedGoogle Scholar
  17. Joldersma, M., Burger, E.H., Semeins, C.M., Klein-Nulend, J. 2000Mechanical stress induces COX-2 mRNA expression in bone cells from elderly womenJ. Biomech.335361PubMedGoogle Scholar
  18. Malchau, H., Herberts, P., Ahnfelt, L. 1993Prognosis of total hip replacement in Sweden. Follow-up of 92,675 operations performed 1978–1990Acta Orthop. Scand.64497506PubMedCrossRefGoogle Scholar
  19. Mankin, H.J., Friedlaender, G.E. 1989Bone and Cartilage Allografts: Physiological and Immunological PrinciplesStock-IncThorofare(NJ)Bone Stock Deficiency in Total Hip Replacement.Google Scholar
  20. Mejdahl, S., Hansen, C.A., Skjodt, H., Reimann, I. 1998Human bone bank allografts stimulate bone resorption and inhibit proliferation in cultures of human osteoblast-like cellsActa Orthop. Scand.696368PubMedCrossRefGoogle Scholar
  21. Murray, D. 1998Surgery and joint replacement for joint diseaseActa Orthop. Scand. Suppl.2811720PubMedGoogle Scholar
  22. Pereira, B.J., Milford, E.L., Kirkman, R.L.,  et al. 1993Low risk of liver disease after tissue transplantation from donors with HCV [letter]Lancet341903904PubMedGoogle Scholar
  23. Robey, P.G., Termine, J.D. 1985Human bone cells in vitroCalcif. Tissue Int.37453460PubMedGoogle Scholar
  24. Schimmel, J.W. 1995Acetabular Reconstruction with Impacted Morsellized Cancellous Bone Grafts in Cemented Revision Hip ArthroplastyNijmegen UniversityNijmegen (The Netherlands)Google Scholar
  25. Schreurs, B.W., Buma, P., Huiskes, R., Slagter, J.L., Slooff, T.J. 1994Morsellized allografts for fixation of the hip prosthesis femoral component. A mechanical and histological study in the goatActa Orthop. Scand.65267275PubMedGoogle Scholar
  26. Shutkin, N.M. 1954Homologous-serum hepatitis following the use of refreigerated bone-bank bone: report of a caseJ. Bone Joint Surg. Am36-A160162PubMedGoogle Scholar
  27. Simonds, R.J., Holmberg, S.D., Hurwitz, R.L. 1992Transmission of human immunodeficiency virus Type 1 from a seronegative organ and tissue donorN. Engl. J. Med.326726732(see comments)PubMedCrossRefGoogle Scholar
  28. T.J. Slooff P. Buma B.W. Schreurs J.W. Schimmel R. Huiskes J. Gardeniers 1996 Acetabular and femoral reconstruction with impacted graft and cement Clin. Orthop. 108 Google Scholar
  29. Slooff, T.J., Schimmel, J.W., Buma, P. 1993Cemented fixation with bone graftsOrthop. Clin. North Am.24667677PubMedGoogle Scholar
  30. Sterck, J.G., Klein-Nulend, J., Lips, P., Burger, E.H. 1998Response of normal and osteoporotic human bone cells to mechanical stress in vitroAm. J. Physiol.274E1113E1120PubMedGoogle Scholar
  31. Sugihara, S., van Ginkel, A.D., Jiya, T.U., van Royen, B.J., van Diest, P.J., Wuisman, P.I. 1999Histopathology of retrieved allografts of the femoral headJ. Bone Joint Surg. Br.81336341PubMedGoogle Scholar
  32. Tagil, M., Aspenberg, P. 1998Impaction of cancellous bone grafts impairs osteoconduction in titanium chambersClin. Orthop.231238Google Scholar
  33. Tomford, W.W. 1995Transmission of disease through transplantation of musculoskeletal allograftsJ. Bone Joint Surg. Am.7717421754PubMedGoogle Scholar
  34. Urist, M.R. 1953Physiologic basis of bone-graft surgery, with special reference to the theory of inductionClin. Orthop.1207216PubMedGoogle Scholar
  35. Yang, R.S., Liu, T.K., Tsai, K.S., Lin-Shiau, S.Y., Lu, K.S. 1992Morphological and immunocytochemical characterization of osteoblast cultures from long bones of neonatal ratsArch. Histol. Cytol.55415422PubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  1. 1.Department of Orthopaedic SurgerySkeletal Tissue Engineering Group, Amsterdam (STEGA)HeerlenThe Netherlands
  2. 2.Department of Oral Cell Biology, Skeletal Tissue Engineering Group, Amsterdam (STEGA)ACTA-Vrije UniversiteitAmsterdamThe Netherlands

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