Even with rehydration, preservation in ethanol influences the mechanical properties of bone and how bone responds to experimental manipulation

Abstract

Typically, bones are harvested at the time of animal euthanasia and stored until mechanical testing. However, storage methods are not standardized, and differential effects on mechanical properties are possible between methods. The goal of this study was to investigate the effects that two common preservation methods (freezing wrapped in saline-soaked gauze and refrigerating ethanol fixed samples) have on bone mechanical properties in the context of an in vitro ribosylation treatment designed to modify mechanical integrity. It was hypothesized that there would be an interactive effect between ribose treatment and preservation method. Tibiae from twenty five 11 week old female C57BL/6 mice were separated into 2 preservation groups. Micro-CT scans of contralateral pairs assessed differences in geometry prior to storage. After 7 weeks of storage, bones in each pair of tibiae were soaked in a solution containing either 0 M or 0.6 M ribose for 1 week prior to 4 point bending tests. There were no differences in any cortical geometric parameters between contralateral tibiae. There was a significant main effect of ethanol fixation on displacement to yield (−16.3%), stiffness (+24.5%), strain to yield (−13.9%), and elastic modulus (+18.5%) relative to frozen specimens. There was a significant main effect of ribose treatment for yield force (+13.9%), ultimate force (+9.2%), work to yield (+22.2%), yield stress (+14.1%), and resilience (+21.9%) relative to control-soaked bones. Postyield displacement, total displacement, postyield work, total work, total strain, and toughness were analyzed separately within each preservation method due to significant interactions. For samples stored frozen, all six properties were lower in the ribose-soaked group (49%–68%) while no significant effects of ribose were observed in ethanol fixed bones. Storage in ethanol likely caused changes to the collagen matrix which prevented or masked the embrittling effects of ribosylation that were seen in samples stored frozen wrapped in saline-soaked gauze. These data illustrate the clear importance of maintaining hydration if the eventual goal is to use bones for mechanical assessments and further show that storage in ethanol can alter potential to detect effects of experimental manipulation (in this case ribosylation).

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“…The fixation method we used potentially limits transferability of the data [ 40 , 41 ]. Although Linde and Sorensen did not find relevant changes in the biomechanical behaviour of human trabecular bone specimens stored in 70% methanol for 100 days [ 42 ] and Mick et al stated no differences for ultimate bending of human cortical specimens after rehydration due to formalin or ethanol-fixation (96%) compared to fresh frozen bones [ 43 ], we do not know the relevance of possible modifications caused by our treatment protocol.…”

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“…Four‐point bending tests were completed with the same femora that were used for micro‐CT scans as described . In brief, bones were placed anterior side down on fixtures that were 18 mm apart.…”

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“…However, because these bones had been stored in 70% ethanol long-term, mechanical testing yielded stiffness values that were increased relative to those generated in testing of the contralateral femoral necks. Because others have reported on this very effect of long-term storage in ethanol 21 , all mechanical property data reported here is from contralateral (right) femora.…”