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

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, fixative agents are commonly used. However, fixatives 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 influence of ethanol and a formalin-based fixative 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 mm3) were obtained from different regions of two fresh human femora (medial, lateral, dorsal, ventral). Specimens were either fixed 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 specific specimen. For statistical analysis a one-way-ANOVA and an LSD post-hoc-test were performed. For ultimate bending strength no significant differences due to formalin or ethanol fixation, as compared to unfixed-fresh bone specimens could be found. And only for few cases significant differences in elastic bending modulus were observed when the two bones were evaluated separately. Since more differences of significant level due to the anatomical region of the samples were determined, the original location seems to have more influence on the evaluated mechanical properties than the method of (chemical) fixation. Consequently, ethanol and the mixed formalin solution can be recommended as a fixation agent for samples in biomechanical testing, if these samples are rinsed in isotonic saline prior to static mechanical testing.