No abstract

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

Mesenchymal stem cells (MSCs) isolated from cadaveric adipose tissue can be obtained in large quantities, and have been reported in the literature to be capable of inducing bone formation in vivo and ex vivo.( 1-6 ) The hypothesis tested whether a demineralized cancellous bone matrix (DCBM) can provide an effective substrate for selection and retention of stem cells derived from the stromal vascular fraction (SVF) of adipose. Human cadaveric adipose tissue was recovered from a donor and digested. The resulting SVF-containing MSCs were seeded onto the demineralized bone allografts, after which the nonadherent cells were washed off. The MSCs were characterized using a flow cytometer and tri-lineage differentiation (osteogenesis, chondrogenesis, and adipogenesis) in vitro. The stem cell-seeded allografts were also characterized for cell number, adherence to the DCBM, osteogenic activity (alkaline phosphatase and Alizarin Red staining), and bone morphorgenic protein (BMP) quantity. Flow cytometry identified a mean total of 7.2% MSCs in SVF and 87.2% MSCs after culture. The stem cells showed the capability of differentiating into bone, cartilage, and fat. On the 21 stem cell-seeded bone allografts, there were consistent, attached, viable cells (100,744±22,762 cells/cube). An assessment of donor age, gender, and body mass index revealed no significant differences in cell numbers. Enzyme-linked immunosorbent assay revealed the presence of BMP-2 and BMP-7. In conclusion, this bone graft contains three key elements for bone regeneration: adhered osteogenic stem cells, 3D osteoconductive bone scaffold, and osteoinductive BMP signal. It therefore has the potential to be effective for bone regeneration.

Gamma radiation is established as a procedure for inactivating bacteria, fungal spores and viruses. Sterilization of soft tissue allografts with high dose (60)Co gamma radiation has been shown to have adverse effects on allograft biomechanical properties. In the current study, bone-patellar tendon-bone (BPTB) allografts from 32 mature sheep were divided into two treatment groups: low-dose radiation at 15 kGy (n = 16) and high-dose radiation at 25 kGy (n = 16) with the contralateral limb serving as a 0 kGy (n = 32) non-irradiated control. Half of the tendons from all treatment groups were biomechanically tested to determine bulk BPTB mechanical properties, cancellous bone compressive properties, and interference screw pull-out strength. The remaining tissues were prepared, implanted, and mechanically tested in an acute in vitro anterior crucial ligament (ACL) reconstruction. Low-dose radiation did not adversely affect mechanical properties of the tendon allograft, bone, or ACL reconstruction compared to internal non-irradiated control. However, high-dose radiation compromised bulk tendon load at failure and ultimate strength by 26.9 and 28.9%, respectively (P < 0.05), but demonstrated no negative effect on the cancellous bone compressive properties or interference screw pull-out strength. Our findings suggest that low dose radiation (15 kGy) does not compromise the mechanical integrity of the allograft tissue, yet high dose radiation (25 kGy) significantly alters the biomechanical integrity of the soft tissue constituent.

This paper describes the validation of an allograft sterilization method specifically designed for the processing methods used at AlloSource in Centennial, CO. The methods used for this validation followed ISO Standard 11137, Method 2B. Three hundred allografts, collected from three defined production batches were dosed using a series of five incremental doses, beginning at 1 kGy and increasing by 1 kGy until 5 kGy was achieved. Following sterilization dosing, each allograft test article was analyzed using a sterility test to identify any viable microorganisms. The number of positive sterility samples was used to calculate the verification dose (1.27 kGy), which was then verified by an additional batch of 100 allografts. The results from this validation indicate that sterility (10(-6) SAL) on human allograft tissue using gamma 60Co radiation can be achieved when a dose of at least 9.2 kGy is employed.