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

• Published in: PloS one 2015-06, Vol.10 (6), p.e0129205-e0129205
• Main Authors: Bernhardt, Anne, Wehrl, Markus, Paul, Birgit, Hochmuth, Thomas, Schumacher, Matthias, Schütz, Kathleen, Gelinsky, Michael
• Format: Article
• Language: English
• Subjects: Acetic anhydride; Acids; Additives; Alginic acid; Analysis; Anhydrides; Bacteria; Biocompatibility; Biological products; Biomaterials; Biomechanics; Biomedical and Dental Materials; Biomedical materials; Biopolymers; Carbon Dioxide; Cold Temperature; Collagen; Collagens; Cylinders; Cytotoxicity; Deactivation; Ethylene; Ethylene oxide; Fungi; Gamma rays; Gels; Gram-negative bacteria; High temperature; Hydrogels; Hydrogen; Hydrogen ion concentration; Hydrogen peroxide; Hygiene; Inactivation; Irradiation; Low temperature; Mechanical properties; Medical electronics; Medical equipment; Mesenchyme; Microorganisms; Morphology; Phages; Polymers; Polysaccharides; Skin & tissue grafts; Spores; Steam; Stem cells; Sterilization; Sterilization - methods; Surgical implants; Temperature effects; Three dimensional models; Transplants & implants; Vegetative cells; Wound healing; Yeast; Yeasts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0129205

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 (scCO₂) treatment is a promising strategy for the terminal sterilization of sensitive biomaterials at low temperature. In combination with low amounts of additives scCO₂ treatment effectively inactivates microorganisms including bacterial spores. We established a scCO₂ 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 scCO₂ 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 scCO₂ treatment. Human mesenchymal stem cell viability and proliferation were not compromised by scCO₂ treatment of these materials and scaffolds. We conclude that scCO₂ 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.