Bone is a nanocomposite biological material with unique mechanical properties, owing to a complex hierarchical structure ranging from the nanoscale up to the macroscale. To better understand bone mechanics, investigation of mechanical properties of different structures on every hierarchical level and the way they interact is a promising approach. Although biomechanical testing of bone at the macroscale has been performed for over a century, investigation of microstructural features, such as individual lamellae, still remains a challenge. Focused ion beam (FIB) milling is an attractive technique for machining bone samples at the microscale, while avoiding inclusions of micro-porosities like lacunae and Haversian canals. So far, microbeams [1][2][3] and micropillars [4][5][6] from animal bone have been mechanically tested under bending with an atomic force microscope (AFM), or under compression with a conventional nanoindenter, respectively, either in dehydrated or rehydrated state. However, micro-scale experiments detailing mechanical properties of human bone have not been reported. Here we present an AFM-based microbeam bending method for micromechanical assessment of human cortical bone in both dehydrated and rehydrated state. FIB-machined microbeams from the femur midshaft of 4 male donors, aged 65-94y were bent with an AFM tip along the beam length [7], first dehydrated in air and then rehydrated in Hank's Balanced Buffer Solution (HBSS). Prior to this, the measurement setup was calibrated by bending FIB-milled Si microbeams of known stiffness utilising an indenter situated within a scanning electron microscope. From the measured stiffness versus bone beam position data, bending moduli were obtained as a fit parameter. Values ranged (25.1-48.7) GPa in air and (7.3-19.1) GPa in HBSS. A decrease of bending modulus up to 5 times was observed for a single microbeam, suggesting a change in deformation mechanism upon rehydration. No significant change of bending moduli was observed with respect to age. Moreover, bending in air exhibited linear elastic behaviour, with same apparent loading and unloading stiffness, whereas in HBSS higher unloading stiffness was observed, suggesting dissipative deformation mechanisms during loading. The dissipated energy in rehydrated samples was calculated as the area between the loading and unloading curve, ranging from 0.093 to 0.655 pJ (i.e. 5.8-64.5% of the total energy) and was found to decrease with age. These preliminary findings suggest important implications of the role of water in the deformation mechanisms of bone, which in turn may play a role in decreased fracture toughness and increased fragility of bone with age.
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