INVESTIGATION OF OXYGEN-DERIVED FREE-RADICAL GENERATION IN CANCELLOUSBONE SPECIMENS OBTAINED FROM DOGS

Generation of free radicals and the ability of various antioxidants to attenuate radical production in freshly procured cancellous bone spec imens was investigated, using spin-trapping and electron spin resonanc e (ESR) techniques. Seven core cancellous bone specimens, 10 mm long a nd 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 alp ha-phenyl-N-tert-butylnitrone in Eagle's minimum essential medium and served as a control. The other 6 specimens from each dog were incubate d in alpha-phenyl-N-tertbutylnitrone/Eagle's minimum essential medium plus 1 of the following antioxidants: superoxide dismutase, catalase, superoxide dismutase/catalase, indomethacin, allopurinol, or deferoxam ine mesylate. All specimens were incubated at: 26 C for 90 minutes, th en frozen at -20 C until they were prepared for analysis by ESR spectr oscopy Each specimen was thawed, homogenized, and extracted in a low-d ielectric 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 a n area: these areas were used to compare intensities of the spin. For each treatment group, the areas from the treated specimens were compar ed with the areas from the control specimens, using a paired t-test. S ignificance was accepted at P less than or equal to 0.05. Spin adducts were detected in all cancellous bone specimens. Specimens incubated i n deferoxamine (P = 0.0017) and superoxide dismutase/catalase (P = 0.0 452) had significantly smaller areas than did control specimens. The a reas 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 atte nuated by in vitro incubation with deferoxamine or superoxide dismutas e/catalase.