Sterilization of three-dimensional tissue engineering scaffolds by supercritical carbon dioxide

Medical devices, implants or patient-care equipment that will come into intimate contact with a patient must be effectively decontaminated to prevent infection or disease transmission. Non-sterile devices have significant ramifications for patient morbidity and mortality and two processes must be undertaken in order to make an item acceptable for use or implantation: cleaning and disinfection (or sterilization) [1]. Sterilization is the destruction of living organisms, and must be done without damaging the material surface and without compromising the bulk material strength or biocompatibility of implantable device. Common sterilization processes include steam autoclaving, gamma irradiation, and chemical treatments with ethylene oxide or hydrogen peroxide plasma. Parameters such as temperature, pressure, energy intensity, sterilizing agent concentration, and cycle time can change widely and the process chemistry and conditions determine its applicability for a given material or device [2]. New sterilization processes continue to be investigated, with emphases on reducing the process temperature and minimizing contamination. An alternative “green” method to classical processes is based on supercritical carbon dioxide (scCO2) technology that is attractive because it’s a non-flammable, non-toxic, physiologically safe, chemically inert and readily available solvent. When heated and compressed above its critical point (7.38 MPa and 304.2 K) CO2 exhibits a liquid-like density but gas-like diffusivity and viscosity and these properties allow to penetrate porous structures easily [3] ; the bactericidal effect is caused by specific interactions between the living cells and the fluid such as cell wall rupture/perforation, inactivation of some key-enzymes, intracellular electrolyte balance perturbation, pH intra/extra cellular variation [4]. We have studied the sterilization of porous PLLA scaffolds for tissue engineering applications in a supercritical fluid extractor. In order to evaluate the efficiency of the sterilization method, the PLLA scaffolds were contaminated with the gram-negative bacteria E. coli. The amount of viable bacteria in each scaffold was estimated by colony-forming unit (CFU). The contaminated scaffolds were treated or not, as control, with carbon dioxide technology. The efficiency of the sterilization treatment was investigated at different value of pressure of CO2, temperature or time. We obtained the total sterilization after just 15 minutes of treatment at the temperature of 37°C and pressure of 150 bar. Differential scanning calorimetry analyses of the so-treated samples did not show significant differences with respect to not-treated samples. As a matter of fact, almost similar melting and crystallization temperatures and enthalpies were measured, thus demonstrating that the crystallinity of the scaffolds was not modified during the treatment. Furthermore, the PLLA scaffolds maintain their biocompatible characteristic after scCO2 sterilization method. In fact the SK-Hep-1 tumor cells grew both on the outer and on the internal surface of the scaffold as demonstrated by a cell viability assay Alamar Blue. [1] N.R. Cholvin, N.R. Bayne, Handbook of Biomaterials Evaluation: Scientific, Technical, and Clinical Testing of Implant Materials, 2nd ed., Macmillan, New York, 1998, pp. 507–522. [2] L.A. Feldman, H.K. Hui, Med. Device Diagn. Ind. Mag., 1997, 57–70. [3] A. Jimenez, G.L. Thompson, M.A. Matthews, T.A. Davis, K. Crocker, J.S. Lyons, A. Trapotsis. J. of Supercritical Fluids, 2007, 42, 366–372. [4] Michel Perrut, J. of Supercritical Fluids, 2012, 66, 359–371. Acknowledgements: the financial support of EU and MIUR for the Project PON01 01287 ‘’SIB’’ is gratefully acknowledged.