Does sterilization with fractionated electron beam irradiation prevent ACL tendon allograft from tissue damage?

PURPOSE: Allografts are frequently used for anterior cruciate ligament (ACL) reconstruction. However, due to the inherent risk of infection, a method that achieves complete sterilization of grafts is warranted without impairing their biomechanical properties. Fractionation of electron beam (FEbeam) irradiation has been shown to maintain similar biomechanical properties compared to fresh-frozen allografts (FFA) in vitro. Therefore, aim of this study was to evaluate the biomechanical properties and early remodelling of grafts that were sterilized with fractionated high-dose electron beam irradiation in an in vivo sheep model. METHODS: ACL reconstruction was performed in 18 mature merino mix sheep. Sixteen were reconstructed with allografts sterilized with FEbeam irradiation (8 × 3.4 kGy) and two with FFA. Eight FFA from prior studies with identical surgical reconstruction and biomechanical and histological analyzes served as controls. Half of the animals were sacrificed at 6 and 12 weeks, and biomechanical testing was performed. Anterior-posterior laxity (APL) was assessed with an AP drawer test at 60° flexion, and load to failure testing was carried out. Histological evaluation of mid-substance samples was performed for descriptive analysis, cell count, crimp and vessel density. For statistical analysis a Kruskal-Wallis test was used for overall group comparison followed by a Mann-Whitney U test for pairwise comparison of the histological and biomechanical parameters. RESULTS: Biomechanical testing showed significantly decreased stiffness in FEbeam compared to FFA at both time points (p ≤ 0.004). APL was increased in FEbeam compared to FFA, which was significant at 6 weeks (p = 0.004). Median of failure loads was decreased in FEbeam grafts, with 12 reconstructions already failing during cyclic loading. Vessel density was decreased in FEbeam compared to FFA at both time points, with significant differences at 12 weeks (p = 0.015). Crimp length was significantly shorter in FEbeam compared to FFA at both time points (p ≤ 0.004) and decreased significantly in both groups from 6 to 12 weeks (p ≤ 0.025). CONCLUSION: ACL reconstruction with fractionated Ebeam sterilization significantly alters the biomechanical properties and the early remodelling process of treated grafts in vivo. Therefore, this sterilization method cannot be recommended for clinical application. As substantial changes in the remodelling are inherent in this study, care in the rehabilitation of even low-dose sterilized allografts, used for ACL reconstruction, is recommended.

High-dose electron beam sterilization of soft-tissue grafts maintains significantly improved biomechanical properties compared to standard gamma treatment.

Allografts have gained increasing popularity in anterior cruciate ligament (ACL) reconstruction. However, one of the major concerns regarding allografts is the possibility of disease transmission. Electron beam (Ebeam) and Gamma radiation have been proven to be successful in sterilization of medical products. In soft tissue sterilization high dosages of gamma irradiation have been shown to be detrimental to biomechanical properties of grafts. Therefore, it was the objective of this study to compare the biomechanical properties of human bone-patellar tendon-bone (BPTB) grafts after ebeam with standard gamma irradiation at medium (25 kGy) and high doses (34 kGy). We hypothesized that the biomechanical properties of Ebeam irradiated grafts would be superior to gamma irradiated grafts. Paired 10 mm-wide human BPTB grafts were harvested from 20 donors split into four groups following irradiation with either gamma or Ebeam (each n = 10): (A) Ebeam 25 kGy, (B) Gamma 25 kGy, (C) Ebeam 34 kGy (D) Gamma 34 kGy and ten non-irradiated BPTB grafts were used as controls. All grafts underwent biomechanical testing which included preconditioning (ten cycles, 0-20 N); cyclic loading (200 cycles, 20-200 N) and a load-to-failure (LTF) test. Stiffness of non-irradiated controls (199.6 ± 59.1 N/mm) and Ebeam sterilized grafts did not significantly differ (152.0 ± 37.0 N/mm; 192.8 ± 58.0 N/mm), while Gamma-irradiated grafts had significantly lower stiffness than controls at both irradiation dosages (25 kGy: 126.1 ± 45.4 N/mm; 34 kGy: 170.6 ± 58.2 N/mm) (p < 0.05). Failure loads at 25 kGy were significantly lower in the gamma group (1,009 ± 400 N), while the failure load was significantly lower in both study groups at high dose irradiation with 34 kGy (Ebeam: 1,139 ± 445 N, Gamma: 1,073 ± 617 N) compared to controls (1,741 ± 304 N) (p < 0.05). Creep was significantly larger in the gamma irradiated groups (25 kGy: 0.96 ± 1.34 mm; 34 kGy: 1.06 ± 0.58 mm) than in the Ebeam (25 kGy: 0.50 ± 0.34 mm; 34 kGy: 0.26 ± 0.24 mm) and control (0.20 ± 0.18 mm) group that did not differ significantly. Strain difference was not different between either control or study groups (controls: 1.0 ± 0.03; Ebeam 34 kGy 1.04 ± 0.018; Gamma 34 kGy 1.0 ± 0.028; 25 kGy: 1.4 ± 2,0; 34 kGy: 1.1 ± 1.1). The most important result of this study was that ebeam irradiation showed significantly less impairment of the biomechanical properties than gamma irradiation. Considering the results of this study and the improved control of irradiation application with electronic beam, this technique might be a promising alternative in soft-tissue sterilization.

Fractionation of high-dose electron beam irradiation of BPTB grafts provides significantly improved viscoelastic and structural properties compared to standard gamma irradiation.

PURPOSE: Irradiation >30 kGy is required to achieve sterility against bacterial and viral pathogens in ACL allograft sterilization. However, doses >20 kGy substantially reduce the structural properties of soft-tissue grafts. Fractionation of irradiation doses is a standard procedure in oncology to reduce tissue damage but has not been applied in tissue graft sterilization. METHODS: Forty-four human 10-mm wide bone-patellar-tendon-bone grafts were randomized into four groups of sterilization with (1) 34 kGy of ebeam (2) 34 kGy gamma (3) 34 kGy fractionated ebeam, and (4) non sterilized controls. Graft´s biomechanical properties were evaluated at time zero. Biomechanical properties were analyzed during cyclic and load-to-failure testing. RESULTS: Fractionation of ebeam irradiation resulted in significantly higher failure loads (1,327 ± 305) than with one-time ebeam irradiation (1,024 ± 204; P = 0.008). Compared to gamma irradiation, significantly lower strain (2.9 ± 1.5 vs. 4.6 ± 2.0; P = 0.008) and smaller cyclic elongation response (0.3 ± 0.2 vs. 0.6 ± 0.4; P = 0.05), as well as higher failure loads (1,327 ± 305 vs. 827 ± 209; P = 0.001), were found. Compared to non-irradiated BPTB grafts, no significant differences were found for any of the biomechanical parameters. Non-irradiated controls had significantly lower cyclic elongation response and higher failure loads than ebeam and gamma irradiation. CONCLUSIONS: In this study, it was found that fractionation of high-dose electron beam irradiation facilitated a significant improvement of viscoelastic and structural properties of BPTB grafts compared to ebeam and gamma irradiation alone, while maintaining levels of non-irradiated controls. Therefore, this technique might pose an important alternative to common methods for sterilization of soft-tissue allografts.