Summary Statement. Supercritical fluid (SCF) sterilization produces clean and osteoconductive allograft bone capable of healing a critical-sised bony defect. SCF treated graft induces an increased anabolic response and decreased catabolic reponse compared to gamma irradiated graft. Introduction. Clinically, allogeneic bone graft is used extensively because it avoids the donor site morbidity associated with autograft. However, there are concerns over the optimal sterilization method to eliminate immunological risks whilst maintaining the biological efficacy of the graft. This study compared the effect of Supercritical fluid (SCF) sterilization and gamma irradiation on the osteoconductivity of allograft bone in a bilateral critical-sised defect rabbit model. Methods. Cortical-cancellous allograft bone was milled, defatted and terminally sterilised with either gamma irradiation at 25kGy or SCF treatment. The graft was then implanted bilaterally into a critical-sised metaphyseal defect in 10 New Zealand White rabbits (n=5 sites per time point per group). Osteoconductivity was evaluated at 2 and 4 weeks to measure the early inflammatory response and early new bone formation respectively, using X-ray, CT, and both qualitative and quantitative histology and immunohistochemistry (Alkaline Phosphatase and Cathepsin-K). Results. Both grafts were well tolerated and osteoconductive. At 2 weeks, there were significant reductions in bone volume and density in the gamma irradiated graft compared to the SCF treated graft as measured by CT. Inside the defect this corresponded with a greater inflammatory response in the gamma irradiated graft, with a less organised fibrous tissue infiltration and mild granuloma reaction. Conversely, the SCF group had a highly organised and densely packed fibrous tissue infiltration around the allograft chips. Immunohistochemistry results supported these findings with an up-regulation in the expression and distribution of Cathepsin-K in the gamma irradiation group; while Alkaline Phosphatase expression was higher in the SCF group. At 4 weeks, resorptive behavior predominated in both groups. Radiographic and CT results detected no significant difference between groups. Histology at 4 weeks showed larger bone chips were undergoing substantial remodeling with areas of simultaneous osteoclastic resorption and osteoblastic new bone formation. Smaller allograft chips and areas of new bone formation were infiltrated by fibrous tissue and undergoing osteoclastic resorption. Quantitative immunohistochemistry showed an up-regulation of Cathepsin-K expression in both groups from 2 to 4 weeks. At both time points Cathepsin-K expression was higher in the gamma irradiated graft compared to the SCF group. This was greatest at 2 weeks where there was a substantial 82% increase in expression which was reduced to a 38% discrepancy at 4 weeks. Alkaline Phosphatase expression was greater in the SCF group at both time-points. Discussion/Conclusion. Allograft bone sterilised with either gamma irradiation or SCF treatment was osteoconductive and capable of healing a critical-sised defect in a rabbit. Gamma irradiated allografts elicited an acute inflammatory reaction when implanted which increased the amount graft resorption compared to the SCF treated bone. Increased osteoclastic resorption may be a concern for
Allograft bone is widely used in orthopaedic surgery, but peri-operative infection of the graft remains a common and disastrous complication. The efficacy of systemic prophylactic antibiotics is unproven, and since the graft is avascular it is likely that levels of antibiotic in the graft are low. Using an electrical potential to accelerate diffusion of antibiotics into allograft bone, high levels were achieved in specimens of both sheep and human allograft. In human bone these ranged from 187.1 mg/kg in endosteal ( Structural allograft can be supplemented directly with antibiotics using iontophoresis. The technique is simple and inexpensive and offers a potential means of reducing the rate of peri-operative infection in allograft surgery. Iontophoresis into allograft bone may also be applicable to other therapeutic compounds.
