Influence of short-term fixation with mixed formalin or ethanol solution on the mechanical properties of human cortical bone

Abstract

Bone specimens obtained for biomechanical experiments are fresh-frozen for storage to slow down tissue degradation and autolysis in long-term storage. Alternatively, due to infectious risks related to the fresh tissues, xative agents are commonly used. However, xatives will likely change the mechanical properties of bone. Existing studies on this issue gave controversial results that are hardly comparable due to a variety of measurement approaches. For this reason, the in uence of ethanol and a formalin-based xative agent was evaluated on the mechanical properties of human cortical bone specimens by means of four-point-bending tests. 127 prismatic specimens with rectangular cross sections (2.5 x 2.5 x 20 mm ) were obtained from di erent regions of two fresh human femora (medial, lateral, dorsal, ventral). Specimens were either xed in ethanol or in a mixed formalin solution or frozen following a given scheme. After two weeks of storage the samples were re-hydrated in isotonic saline and subsequently tested mechanically. The elastic bending modulus and ultimate bending strength were computed considering the actual dimensions of each speci c specimen. For statistical analysis a one-way-ANOVA and an LSD post-hoc-test were performed. For ultimate bending strength no signi cant di erences due to formalin or ethanol xation, as compared to un xed-fresh bone specimens could be found. And only for few cases signicant di erences in elastic bending modulus were observed Rostock, Germany, E-mail: marko.schulze@med.uni-rostock.de when the two bones were evaluated separately. Since more di erences of signi cant level due to the anatomical region of the samples were determined, the original location seems to have more in uence on the evaluated mechanical properties than the method of (chemical) xation. Consequently, ethanol and the mixed formalin solution can be recommended as a xation agent for samples in biomechanical testing, if these samples are rinsed in isotonic saline prior to static mechanical testing.

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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. Anyway, as the comparison between the groups is more significant than the absolute values and all bones were treated equally, this systematic failure should not affect the main deductions drawn by our study.…”

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“…The storage period in 70% ethanol was less than a month. Short-term storage in 70% ethanol has been shown to have no negative effects on the mechanical properties [29,30]. Prior to mechanical testing, femurs were rehydrated in saline for 24 h at 4°C.…”

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

“…However, these postyield properties demonstrated significant interaction effects which necessitated separate analyses within each preservation group. Previous studies have indicated that the negative effects of soaking in ethanol can be reversed with rehydration prior to testing in compression [6], tension [7], and beam bending [8]. However, one of these three studies only reported preyield parameters [6], and the postyield properties reported in the other two were limited to ultimate stress [8] or ultimate stress and strain to failure [7].…”

“…Previous studies have indicated that the negative effects of soaking in ethanol can be reversed with rehydration prior to testing in compression [6], tension [7], and beam bending [8]. However, one of these three studies only reported preyield parameters [6], and the postyield properties reported in the other two were limited to ultimate stress [8] or ultimate stress and strain to failure [7]. Although the one study reporting strain to failure did not show a significant difference [7], the sample size was low which necessitated non-parametric statistical analyses and a loss of power as indicated by the authors.…”

“…Some labs store bones in ethanol at 4 °C and then rehydrate prior to testing [5]. Studies in the literature suggest that rehydrating the bone prior to testing allows its mechanical properties to recover to normal values [6–8], but rigorous testing of this question has not been undertaken. It is important to understand what effects, if any, different storage methods have on bone properties including stiffness, strength, and ductility in order to increase reproducibility across labs.…”