Investigation of oxygen-derived free radical generation in cancellous bone specimens obtained from dogs

Generation of free radicals and the ability of various antioxidants to attenuate radical production in freshly procured cancellous bone specimens was investigated, using spin-trapping and electron spin resonance (ESR) techniques. Seven core cancellous bone specimens, 10 mm long and 7.9 mm in diameter, were obtained using aseptic technique, from the proximal portion of the humerus of 9 adult mixed-breed dogs. One core cancellous bone specimen from each dog was incubated in spin trap alpha-phenyl-N-tert-butylnitrone in Eagle's minimum essential medium and served as a control. The other 6 specimens from each dog were incubated in alpha-phenyl-N-tert-butylnitrone/Eagle's minimum essential medium plus 1 of the following antioxidants: superoxide dismutase, catalase, superoxide dismutase/catalase, indomethacin, allopurinol, or deferoxamine mesylate. All specimens were incubated at 26 C for 90 minutes, then frozen at -20 C until they were prepared for analysis by ESR spectroscopy. Each specimen was thawed, homogenized, and extracted in a low-dielectric organic solvent prior to obtaining an ESR spectrum which was analyzed for hyperfine splitting constants to identify radicals. Each first-derivative spectrum was digitally double-integrated to obtain an area: these areas were used to compare intensities of the spin. For each treatment group, the areas from the treated specimens were compared with the areas from the control specimens, using a paired t-test. Significance was accepted at P less than or equal to 0.05. Spin adducts were detected in all cancellous bone specimens. Specimens incubated in deferoxamine (P = 0.0017) and superoxide dismutase/catalase (P = 0.0452) had significantly smaller areas than did control specimens. The areas for the other treatment groups did not differ significantly from controls. Our results substantiate free radical production in freshly procured cancellous bone specimens and that radical formation is attenuated by in vitro incubation with deferoxamine or superoxide dismutase/catalase.