Circumferential Arc-Shaped Microcracks in Haversian Bone: Lead-Uranyl Acetate Staining for Micro-CT Imaging
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Circumferential Arc-Shaped Microcracks in Haversian Bone: Lead-Uranyl Acetate Staining for Micro-CT Imaging Vincent Ebacher, Rizhi Wang Department of Materials Engineering, University of British Columbia, 309-6350 Stores Road, Vancouver, BC, Canada, V6T 1Z4. ABSTRACT Despite much progress in recent years, the nature of microcracking in bone at the nanometer scale is still not well understood. This is partly due to the complexity of bone's hierarchical structure, but also to the difficulty of detecting cracks at very fine scales. Bone microcracking is typically detected using fluorescent dye staining techniques followed by optical or laser microscopy examinations. However, fluorescence-based methods are limited to sub-micron resolution and do not fit three-dimensional imaging such as micro-CT or high resolution imaging such as electron microscopy. This pilot study explores the potential of a heavy metal staining technique to label nano-sized cracks in bone that could be detected by electron microscopy and, albeit at a larger scale, by micro-computed tomography. Upon further development, the method described here may lead to the nano-meter scale characterization of bone microcracking. INTRODUCTION The resistance to fracture of most engineering materials is mainly due to their ability to dissipate energy through different deformation mechanisms taking place at the yield and postyield stages. In the case of bone, microcracking is such a mechanism associated with its inelastic deformation [1]. Although much progress has been made in recent years [2-5], the nature of microcracking and its relation to bone's hierarchical structure down to the nano-meter level is poorly understood. This is partly due to the complexity of bone's ultrastructure, but also due to the difficulty of detecting cracks at very fine scales. Typically, bone microcracking is detected via fluorescence microscopy which involves a staining procedure where fluorescent agents are used to label the microcracks. This technique offers several advantages. First, it allows us to distinguish deformation-induced microcracks from other artifacts introduced during preparation. Second, it stains not only surface microcracks but also bulk microcracks, thus providing a more general representation of the microcracking. Third, when combined with laser scanning confocal microscopy (LSCM), it offers threedimensional imaging capability [6,7]. However, since the technique is based on optical microscopy, its resolution is limited to sub-micron sized features. The combination of high resolution micro-computed tomography (micro-CT) and electron microscopy (EM) could solve the resolution issue while offering comparable and even additional advantages to fluorescencebased methods, provided that the microcracks labeling agent is detectable by these techniques. Commonly used for biological tissues [8], lead-uranyl acetate staining has been applied to the detection of bone microdamage. Based on the principles of bulk staining with basic fuchsin [9], Schaffler et al. [10
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