In this work the sterilization with dense carbon dioxide of poly(L-lactic acid) (PLLA) porous scaffolds intended for tissue engineering applications was investigated with the main objective of confirming the three-dimensional efficacy of the treatment and of analysing the scaffold properties after CO2 treatment. For this purpose the scaffold was contaminated with a conventional bacterium (Escherichia coli) and with spores (Streptomyces coelicolor), a species more fascinating and difficult to inactivate. Contamination was performed in such a way to soak the whole matrix with bacteria and spores. The effect of pressure and treatment time on the efficacy of the sterilization was evaluated. The E. coli was eradicated from the whole matrix after just 5 min of exposure to supercritical CO2 at 10 MPa and 40 degrees C. The spores required more severe conditions due to their different and very resistant structures since complete inactivation was obtained after 360 min of exposure at 30 MPa and 40 degrees C or at 30 MPa and 30 degrees C after the addition of 200 ppm of H2O2 to near critical CO2. The treatment with dense CO2 did not alter the biocompatibility and the structure of the scaffold as demonstrated by biological culture tests and calorimetric and SEM analyses. Collected data suggest that dense CO2 is a promising alternative to conventional sterilization techniques to sterilize biodegradable PLLA scaffolds. (C) 2016 Elsevier B.V. All rights reserved.