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Abstract
In order to assess the influence of eight different sterilisation and disinfection methods for bone allografts on adhesion, proliferation, and differentiation of human bone marrow stromal cells (BMSC), cells were grown in culture and then plated onto pieces of human bone allografts. Following processing methods were tested: autoclavation (AUT), low-temperature-plasma sterilisation of demineralised allografts (D-LTP), ethylene oxide sterilisation (EtO), fresh frozen bone (FFB), 80 °C-thermodisinfection (80°C), γ-irradiation (Gamma), chemical solvent disinfection (CSD), and Barrycidal®-disinfection (BAR). The seeding efficiency was determined after one hour to detect the number of attached cells before mitosis started. The cell viability was determined after 3, 7, and 21 days. Tests to confirm the osteoblastic differentiation included histochemical alkaline phosphatase staining and RT-PCR for osteocalcin.
Human BMSC showed greatest attachment affinities for D-LTP-, 80°C-, and CSD-allografts, whereas less cells were found attached to AUT-, EtO-, FFB-, Gamma-, and BAR-probes. Cell viability assays at day 3 revealed highest proliferation rates within the FFB- and 80°C-groups, whereas after 21 days most viable cells were found in D-LTP-, 80°C-, CSD-, and Gamma-groups. BAR-treatment showed a considerably toxic effect and therefore was excluded from all further experiments. Highest AP-activity and gene expression of osteocalcin were detected in the D-LTP-group in comparison with all other groups.
In summary, our results demonstrate that cell adhesion, final population, and function of BMSC are influenced by different disinfection and sterilisation methods. Therefore, processing-related alterations of BMSC-function may be important for the success of bone grafting. The experimental setup used in the present work may be useful for further optimisation of bone allograft processing.
© 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
Keywords: Bone allograft; Osteoblast; Disinfection; Sterilisation; Transplantation
Introduction
Bone grafting is frequently necessary to fill bone defects and gaps in multifragmentary fractures, nonunions, cysts, or in defects resulting from inflammatory or tumourous skeletal diseases. Bone grafts must conform to certain basic requirements, amongst which biomechanical, osteoconductive, osteoinductive, or microbiological traits are equally important. In order to reduce the host immune reaction and the risk of bacterial and viral transmission bone allograft preparations are cleaned and processed using different protocols [4,6]. Osteoinductive, osteoconductive, and biomechanical properties of different allograft preparations vary depend-ing on methods of graft processing [4,6]. Knowledge about direct effects...