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

Abstract

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.

Biomaterial characterization of off-the-shelf decellularized porcine pericardial tissue for use in prosthetic valvular applications

“…Although decellularization shifted the mechanical properties of the affected pericardium towards those of the native valvular cusps, sterilization had a moderate effect in the opposing direction. Although the major method of sterilization, via sCO 2 , has been reported to not have a cross‐linking effect on biomaterials (Bernhardt et al, ; Hennessy et al, ), interesting compressive and tensile increases post‐treatment have been noted in studies utilizing this technique, as well as in the data at hand. Bernhardt et al () postulated that the high pressure utilized with sCO 2 sterilization may impact biomaterial nanostructure in unexpected manners.…”

Biologic Scaffolds

“…ETO can react with the free amine groups of collagen, affecting the mechanical properties of ECM bioscaffolds, and high-energy radiation damages protein chains and promotes the formation of free radicals (Proffen et al 2015). Supercritical CO 2 sterilization 10 has been investigated for sterilization of collagen scaffolds and showed minimal compromise of mechanical properties (Bernhardt et al 2015).…”

Mechanism of Bacillus subtilis spore inactivation by and resistance to supercritical CO2 plus peracetic acid

“…; Bernhardt et al . ). Other variations on scCO 2 have also been tested for inactivating spores of Bacillus species, including microbubbles of scCO 2 and scCO 2 treatment following a pulsed electric field treatment that sensitizes spores to scCO 2 (Ishikawa et al .…”

“…,b; Bernhardt et al . ), conditions that alone are not especially damaging to drugs or devices. However, while such conditions can be efficient at inactivating growing bacteria of a variety of species, generally higher temperatures or pressures are needed to efficiently inactivate spores (Ishikawa et al .…”