Ribose pre-treatment can protect the fatigue life of γ-irradiation sterilized bone.

Structural bone allografts are often sterilized with γ-irradiation to decrease infection risk, which unfortunately degrades the bone collagen connectivity, making the bone weak and brittle. In previous studies, we successfully protected the quasi-static mechanical properties of human cortical bone by pre-treating with ribose, prior to irradiation. This study focused on the quasi-static and fatigue tensile properties of ribose treated irradiated sterilized bone allografts. Seventy-five samples were cut from the mid-shaft diaphysis of human femurs into standardized dog-bone shape geometries for quasi-static and fatigue tensile testing. Specimens were prepared in sets of three adjacent specimens. Each set was made of a normal (N), irradiated (I) and ribose pre-treated + irradiation (R) group. The R group was incubated in a 1.2 M ribose solution before γ-irradiation. The quasi-static tensile and decalcified tests were conducted to failure under displacement control. The fatigue samples were tested under cyclic loading (10 Hz, peak stress of 45MP, minimum-to-maximum stress ratio of 0.1) until failure or reaching 10 million cycles. Ribose pre-treatment significantly improved significantly the mechanical properties of irradiation sterilized human bone in the quasi-static tensile and decalcified tests. The fatigue life of the irradiated group was impaired by 99% in comparison to the normal control. Surprisingly, the R-group has significantly superior properties over the I-group and N-group (p < 0.01, p < 0.05) (> 100%). This study shows that incubating human cortical bone in a ribose solution prior to irradiation can indeed improve the fatigue life of irradiation-sterilized cortical bone allografts.

Pre-clinical evaluation of bone allograft toughened with a novel sterilization method: An in vivo rabbit study.

Bone allografts often undergo γ-irradiation sterilization to decrease infection risk. However this consequently degrades bone collagen and makes the allograft brittle. Our laboratory has previously found that pre-treatment with ribose ex vivo protects the bone. However, it remains unclear whether or not ribose-treated γ-irradiated allografts are able to unite and remodel in vivo. Using New Zealand White rabbits (NZWr), we aimed to evaluate if ribose-treated allografts can unite with host bone (compared to untreated (fresh-frozen) and conventionally-irradiated allografts). A critically-sized defect was created in the radii of NZWr and reconstructed with allografts fixed with an intramedullary Kirschner wire. Healing and union were assessed at 2, 6, and 12 weeks post operation, with radiographs, µCT, static and dynamic histomorphometry, backscatter electron microscopy, and torsion testing. Intramedullary fixation achieved stable reconstructions and bony union in all groups and no differences were found in the radiographic and biomechanical parameters tested. Interestingly, γ-irradiated allografts had significantly less bone volume due to evident resorption of the grafts. In contrast, ribose pre-treatment protected γ-irradiated allografts from this bone loss, with results similar to the fresh frozen controls. In conclusion, ribose-pretreated γ-irradiated allografts were able to unite in vivo. In addition to achieving bony union with host bone, ribose pre-treatment may protect against allograft resorption. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

The effect of ribose pre-treatment of cortical bone on γ-irradiation sterilization effectiveness.

Reconstruction of large skeletal defects is a significant and challenging issue. Tissue banks often use γ-irradiation (15-35 kGy) to sterilize bone allografts, which, however, drastically impairs the post-yield mechanical properties. In previous studies, we reported the development of a method that protects human bone collagen connectivity through ribose crosslinking while still undergoing γ-irradiation. Given these promising results, the next step was to determine if the presence of ribose within the bone tissue would interfere with the effectiveness of the γ-irradiation sterilization against bacteria. This study had two stages. The aim of the first stage was to assess the protective effect of ribose in solution using a Bacillus pumilus spore strip model. The aim of the second stage was to assess the protective effect of ribose (R) on spores within a human cortical bone model in comparison to conventionally irradiated bone (I). Treatment of B. pumilus spore strips with ribose in solution led to temperature-dependent effects on spore viability versus spore strips treated with PBS alone. Ribose solution at 60 °C led to a notable two logs decrease in spore count relative to PBS at 60 °C. In the human bone model, the number of spores in the I and R groups were greatly decreased in comparison to the non-irradiated N group. No spore colonies were detected in the R group (n = 4) whereas two of the four plates of group I formed colonies. This study provides evidence that the method of pre-treating bone with ribose crosslinking prior to irradiation sterilization, while improving irradiation sterilized bone allograft quality, also may improve the effectiveness of the sterilization process.

Development of a novel method for the strengthening and toughening of irradiation-sterilized bone allografts.

Reconstruction of large skeletal defects is a significant and challenging issue. Bone allografts are often used for such reconstructions. However, sterilizing bone allografts by using γ-irradiation, damages collagen and causes the bone to become weak, brittle and less fatigue resistant. In a previous study, we successfully protected the mechanical properties of human cortical bone by conducting a pre-treatment with ribose, a natural and biocompatible agent. This study focuses on examining possible mechanisms by which ribose might protect the bone. We examined the mechanical properties, crosslinking, connectivity and free radical scavenging potentials of the ribose treatment. Human cortical bone beams were treated with varying concentration of ribose (0.06-1.2 M) and γ-irradiation before testing them in 3-point bending. The connectivity and amounts of crosslinking were determined with Hydrothermal-Isometric-Tension testing and High-Performance-Liquid-Chromatography, respectively. The free radical content was measured using Electron Paramagnetic Resonance. Ribose pre-treatment improved the mechanical properties of irradiation sterilized human bone in a pre-treatment concentration-dependent manner. The 1.2 M pre-treatment provided >100% of ultimate strength of normal controls and protected 76% of the work-to-fracture (toughness) lost in the irradiated controls. Similarly, the ribose pre-treatment improved the thermo-mechanical properties of irradiation-sterilized human bone collagen in a concentration-dependent manner. Greater free radical content and pentosidine content were modified in the ribose treated bone. This study shows that the mechanical properties of irradiation-sterilized cortical bone allografts can be protected by incubating the bone in a ribose solution prior to irradiation.

Biomechanical comparison of the human cadaveric pelvis with a fourth generation composite model.

The use of cadavers for orthopaedic biomechanics research is well established, but presents difficulties to researchers in terms of cost, biosafety, availability, and ease of use. High fidelity composite models of human bone have been developed for use in biomechanical studies. While several studies have utilized composite models of the human pelvis for testing orthopaedic reconstruction techniques, few biomechanical comparisons of the properties of cadaveric and composite pelves exist. The aim of this study was to compare the mechanical properties of cadaveric pelves to those of the 4th generation composite model. An Instron ElectroPuls E10000 mechanical testing machine was used to load specimens with orientation, boundary conditions and degrees of freedom that approximated those occurring during the single legged phase of walking, including hip abductor force. Each specimen was instrumented with strain gauge rosettes. Overall specimen stiffness and principal strains were calculated from the test data. Composite specimens showed significantly higher overall stiffness and slightly less overall variability between specimens (composite K=1448±54N/m, cadaver K=832±62N/m; p<0.0001). Strains measured at specific sites in the composite models and cadavers were similar (but did differ) only when the applied load was scaled to overall construct stiffness. This finding regarding strain distribution and the difference in overall stiffness must be accounted for when using these composite models for biomechanics research. Altering the cortical wall thickness or tuning the elastic moduli of the composite material may improve future generations of the composite model.

γ-Irradiation sterilized bone strengthened and toughened by ribose pre-treatment.

OBJECTIVE: This study tested the hypothesis that a ribose-based pre-treatment would protect the strength, ductility and toughness of γ-irradiation sterilized cortical bone. METHODS: Experiment 1: The effects of ribose pre-treatment (1.8M in PBS at 60°C for 24h) prior to 33 kGy of irradiation on strength, ductility and toughness (beams in three-point bending) and fracture toughness (J-integral at instability in single edge notched (bending)) were tested against matched non-irradiated and irradiated controls from bovine tibiae. Experiment 2: Three-point bending tests were conducted using beams from human femora (males, 59-67 years). Bone collagen thermal stability and network connectivity were examined using hydrothermal isometric tension testing. RESULTS: Ribose pre-treatment protected the strength, ductility and toughness of irradiation sterilized bovine and human specimens to differing degrees. Their ultimate strength was not detectably different from non-irradiated control levels; toughness in bovine and human specimens was protected by 57 and 76%, respectively. Untreated human bone was less affected by irradiation and ribose pre-treatment was more effective in human bone than bovine bone. CONCLUSIONS: This paper presents the first proof-of-principle that irradiation-sterilized bone with improved mechanical properties can be produced through the application of a ribose pre-irradiation treatment, which provides a more stable and connected collagen network than found in conventionally irradiated controls.