Ethanol and formaldehyde fixation irreversibly alter bones' organic matrix

Abstract

Introduction: Biomechanical tests on bones are frequently accomplished in anatomically fixed tissues. The effects of ethanol or formaldehyde based fixation in bone material properties are subject to controversial discussions, regarding their appropriateness and usability to answer clinical questioning or biomechanical issues. We hypothesized that ethanol and formaldehyde irreversibly change bone material properties, and that this effect is mainly related to the bone's organic matrix.

Material and methods: Fixation related alterations in material properties were investigated in six fresh and two macerated human coxal bones by means of three-dimensional laser vibrometry based modal analysis. Ethanol or formaldehyde fixation were performed in one macerated and three unfixed specimens each. Changes in specimen weight and modal frequencies related to fixation, rinsing and drying were obtained. Modal assurance criterion (MAC) values were recorded to determine altered bone anisotropy.

Results: Due to fixation, modal frequencies were irreversibly altered in unfixed specimens, indicating weight loss in ethanol and structural changes in formaldehyde fixed specimens. In the macerated and inorganic controls, fixation related weight and modal frequency changes were reversible by rinsing. In the unfixed specimens, bone anisotropy was irreversibly altered by both modes of fixation, whereas the fixation related changes in bony anisotropy were reversible in the macerated controls after rinsing.

Discussion: Anatomical fixation that includes ethanol or formaldehyde irreversibly alters material properties of unfixed bones and impacts bone anisotropic properties, caused by changes in the organic matrix. In macerated bones that exclusively consisted of inorganic mineral salts, the observed effects on material properties and anisotropy were reversible. Conclusively, anatomical fixation on basis of ethanol or formaldehyde cannot be recommended, if material characteristics close to the vital state are of interest. Modal analysis is a potential method to gain insight into material properties, revealing the influence of the organic bone matrix on coxal bone elasticity.

Keywords: Bone anisotropy; Ethanol fixation; Formaldehyde fixation; MAC; Modal analysis; Modal assurance criterion..