Bone embrittlement and collagen modifications due to high-dose gamma-irradiation sterilization.
Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness. Using cortical bone from bovine tibiae, sample beams irradiated at 33kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture. Irradiation resulted in a 62% loss of work-to-fracture (p≤0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For DSC, there was a 20% decrease in thermal stability (p<0.001) with a 100% increase (p<0.001) in enthalpy of denaturation (melting). HIT testing also showed a decrease in thermal stability (20% lower denaturation temperature, p<0.001) and greatly reduced measures of collagen network connectivity (p<0.001). Interestingly, the increase in enthalpy of denaturation suggests that irradiated collagen requires more energy to denature (melt), perhaps a result of alterations in the hydrogen bonding sites (increased carbonyl content detected in the insoluble collagen) on the irradiated bone collagen. Altogether, this new data strongly indicates that a large loss of overall collagen connectivity due to collagen fragmentation resulting from γ-irradiation sterilization leads to inferior cortical bone toughness. In addition, notable changes in the thermal denaturation of the bone collagen along with chemical indicators of oxidative modification of the bone collagen indicate that the embrittlement may be a function not only of collagen fragmentation but also of changes in bonding.
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 Collagen Street, Toronto, ON M5S 3G9, Canada; Musculoskeletal Research Laboratory, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 25 Orde Street, Room 417, Toronto, ON M5T 3H7, Canada.
Brianne
B
Burton
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 Collagen Street, Toronto, ON M5S 3G9, Canada; Musculoskeletal Research Laboratory, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 25 Orde Street, Room 417, Toronto, ON M5T 3H7, Canada.
Anne
A
Gaspar
Musculoskeletal Research Laboratory, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 25 Orde Street, Room 417, Toronto, ON M5T 3H7, Canada.
David
D
Josey
Musculoskeletal Research Laboratory, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 25 Orde Street, Room 417, Toronto, ON M5T 3H7, Canada.
Jindra
J
Tupy
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 Collagen Street, Toronto, ON M5S 3G9, Canada; Musculoskeletal Research Laboratory, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 25 Orde Street, Room 417, Toronto, ON M5T 3H7, Canada; Department of Material Science, University of Toronto, 184 College Street, Toronto, ON M5S 3E4, Canada.
Marc D
MD
Grynpas
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 Collagen Street, Toronto, ON M5S 3G9, Canada; Musculoskeletal Research Laboratory, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 25 Orde Street, Room 417, Toronto, ON M5T 3H7, Canada; Department of Material Science, University of Toronto, 184 College Street, Toronto, ON M5S 3E4, Canada; Department of Surgery, University of Toronto, 149 College Street, Toronto, ON M5T 1P5, Canada; Division of Orthopaedic Surgery, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada. Electronic address: willett@lunenfeld.ca.
Thomas L
TL
Willett
D064591:Allografts; D000818:Animals; D015519:Bone Density / Q000528:radiation effects; D016025:Bone Transplantation / Q000379:methods*; D001842:Bone and Bones / Q000528:radiation effects*; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D019245:Compressive Strength / Q000528:radiation effects*; D004591:Electrophoresis, Polyacrylamide Gel; D005720:Gamma Rays / Q000009:adverse effects*; D013242:Sterilization / Q000379:methods*; D013314:Stress, Mechanical
D003094:Collagen
Collagen; Cortical bone; Irradiation; Sterilization; Toughness