Effect of gamma irradiation and supercritical carbon dioxide sterilization with Novakill™ or ethanol on the fracture toughness of cortical bone

Abstract

Sterilization of structural bone allografts is a critical process prior to their clinical use in large cortical bone defects. Gamma irradiation protocols are known to affect tissue integrity in a dose dependent manner. Alternative sterilization treatments, such as supercritical carbon dioxide (SCCO₂ ), are gaining popularity due to advantages such as minimal exposure to denaturants, the lack of toxic residues, superior tissue penetration, and minor impacts on mechanical properties including strength and stiffness. The impact of SCCO₂ on the fracture toughness of bone tissue, however, remains unknown. Here, we evaluate crack initiation and growth toughness after 2, 6, and 24 h SCCO₂ -treatment using Novakill™ and ethanol as additives on ~11 samples per group obtained from a pair of femur diaphyses of a canine. All mechanical testing was performed at ambient air after 24 h soaking in Hanks' balanced salt solution (HBSS). Results show no statistically significant difference in the failure characteristics of the Novakill™-treated groups whereas crack growth toughness after 6 and 24 h of treatment with ethanol significantly increases by 37% (p = .010) and 34% (p = .038), respectively, compared to an untreated control group. In contrast, standard 25 kGy gamma irradiation causes significantly reduced crack growth resistance by 40% (p = .007) compared to untreated bone. FTIR vibrational spectroscopy, conducted after testing, reveals a consistent trend of statistically significant differences (p < .001) with fracture toughness. These trends align with variations in the ratios of enzymatic mature to immature crosslinks in the collagen structure, suggesting a potential association with fracture toughness. Additional Raman spectroscopy after testing shows a similar trend with statistically significant differences (p < .005), which further supports that collagen structural changes occur in the SCF-treated groups with ethanol after 6 and 24 h. Our work reveals the benefits of SCCO₂ sterilization compared to gamma irradiation.

Keywords: allograft sterilization; bone; crack resistance curve behavior; deformation and fracture toughness; mechanical characterization; supercritical fluid.