Improved Sterilization of Sensitive Biomaterials with Supercritical Carbon Dioxide at Low Temperature

Anne Bernhardt*, Markus Wehrl, Birgit Paul, Thomas Hochmuth, Matthias Schumacher, Kathleen Schuetz, Michael Gelinsky

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


The development of bio-resorbable implant materials is rapidly going on. Sterilization of those materials is inevitable to assure the hygienic requirements for critical medical devices according to the medical device directive (MDD, 93/42/EG). Biopolymer-containing biomaterials are often highly sensitive towards classical sterilization procedures like steam, ethylene oxide treatment or gamma irradiation. Supercritical CO2 (scCO(2)) treatment is a promising strategy for the terminal sterilization of sensitive biomaterials at low temperature. In combination with low amounts of additives scCO(2) treatment effectively inactivates microorganisms including bacterial spores. We established a scCO(2) sterilization procedure under addition of 0.25% water, 0.15% hydrogen peroxide and 0.5% acetic anhydride. The procedure was successfully tested for the inactivation of a wide panel of microorganisms including endospores of different bacterial species, vegetative cells of gram positive and negative bacteria including mycobacteria, fungi including yeast, and bacteriophages. For robust testing of the sterilization effect with regard to later application of implant materials sterilization all microorganisms were embedded in alginate/agarose cylinders that were used as Process Challenge Devices (PCD). These PCD served as surrogate models for bioresorbable 3D scaffolds. Furthermore, the impact of scCO(2) sterilization on mechanical properties of polysaccharide-based hydrogels and collagen-based scaffolds was analyzed. The procedure was shown to be less compromising on mechanical and rheological properties compared to established low-temperature sterilization methods like gamma irradiation and ethylene oxide exposure as well as conventional steam sterilization. Cytocompatibility of alginate gels and scaffolds from mineralized collagen was compared after sterilization with ethylene oxide, gamma irradiation, steam sterilization and scCO(2) treatment. Human mesenchymal stem cell viability and proliferation were not compromised by scCO(2) treatment of these materials and scaffolds. We conclude that scCO(2) sterilization under addition of water, hydrogen peroxide and acetic anhydride is a very effective, gentle, non-cytotoxic and thus a promising alternative sterilization method especially for biomaterials.
Original languageEnglish
Article numbere0129205
Issue number6
Publication statusPublished - 2015
Externally publishedYes

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