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Screening and processing methods currently in place have made the risk of bacterial and viral infections from allograft tissues extremely low. However, the development of a terminal sterilization method that does not adversely affect tissue function would provide an added safety to tissues for transplantation. We assessed whether high-dose gamma irradiation could be used as an effective terminal sterilization method for allografts without impairing the preimplantation mechanical integrity of the tissues. Semitendinosus tendons were pretreated with a radioprotectant solution and then irradiated to 50 kGy under well-defined conditions that included a tight dose range and maintained low temperatures. Maximum force, strain, stress, modulus, and strain energy density for tendons irradiated to 50 kGy were compared to nonirradiated control tendons and tendons irradiated to 18 kGy by a commercial tissue bank using their existing method. The preimplantation biomechanical properties of the 50-kGy group compared favorably to the nonirradiated and 18 kGy groups. A study to evaluate the postimplantation mechanical and biological performance of grafts irradiated to 50 kGy is ongoing. Pathogen inactivation was also quantified following 50 kGy of irradiation, with !4.5 logs of Sindbis virus and 4.9 logs of parvovirus kill achieved. Analysis of Clostridium sordellii inactivation kinetics indicated that a 16 log 10 reduction is predicted with 50 kGy of irradiation. A high dose of gamma irradiation using the described conditions can reduce infectious risks associated with soft tissue allografts while maintaining the preimplantation biomechanical performance of the tissues. ß

The ability of Porphyromonas gingivalis to acquire iron in the iron-limited environment of the host is crucial to the colonization of this organism. We report here on the isolation and characterization of a transpositional insertion mutant of P. gingivalis A7436 (designated MSM-3) which is defective in the utilization and transport of hemin. P. gingivalis MSM-3 was selected on the basis of its nonpigmented phenotype on anaerobic blood agar following mutagenesis with the Bacteroides fragilis transposon Tn4351. P. gingivalis MSM-3 grew poorly when supplied with hemin as a sole source of iron; however, growth was observed with hemoglobin or inorganic iron. P. gingivalis MSM-3 grown in either hemin-replete or hemin-depleted conditions bound and transported less [ 14 C]hemin or [ 59 Fe]hemin than did the parent strain. At 4 h, P. gingivalis MSM-3 grown in hemin-replete conditions transported only 10,000 pmol of hemin per mg of protein, or 14% of the amount transported by P. gingivalis A7436. Unlike P. gingivalis A7436, hemin binding and transport by P. gingivalis MSM-3 were not tightly regulated by hemin or iron. Examination of P. gingivalis MSM-3 cultures by electron microscopy revealed an overproduction of membrane vesicles, and determination of the dry weight of purified vesicles indicated that P. gingivalis MSM-3 produced twice as much membrane vesicles as did strain A7436. Extracellular vesicles isolated from P. gingivalis MSM-3 also were found to express increased hemolytic and trypsin-like protease activities compared with the parent strain. When inoculated into subcutaneous chambers implanted in mice, P. gingivalis MSM-3 was highly infectious and more invasive than the parent strain, as indicated by secondary lesion formation and death. Taken together, these results indicate that the decreased transport of hemin by P. gingivalis MSM-3 results in the increased expression of several virulence factors which may be coordinately regulated by hemin.