Nanoindentation of bone: Comparison of specimens tested in liquid and embedded in polymethylmethacrylate

Elastic modulus of bone was investigated by nanoindentation using common methods of sample preparation, data collection, and analysis, and compared to dynamic mechanical analysis (DMA: three-point bending) for the same samples. Nanoindentation (Berkovich, 5 m and 21 m radii spherical indenters) and DMA were performed on eight wet and dehydrated (100% ethanol), machined equine cortical bone beams. Samples were embedded in polymethylmethacrylate (PMMA) and mechanical tests repeated. Indentation direction was transverse to the bone long axis while DMA tested longitudinally, giving approximately 12% greater modulus in DMA. For wet samples, nanoindentation with spherical indenters revealed a low modulus surface layer. Estimates of the volume of material contributing to elastic modulus measurement showed that the surface layer influences the measured modulus at low loads. Consistent results were obtained for embedded tissue regardless of indenter geometry, provided appropriate methods and analysis were used. Modulus increased for nanoindentation (21 m radius indenter) from 11.7 GPa ± 1.7 to 15.0 GPa ± 2.2 to 19.4 GPa ± 2.1, for wet, dehydrated in ethanol, and embedded conditions, respectively. The large increases in elastic modulus caused by replacing water with ethanol and ethanol with PMMA demonstrate that the role of water in fine pore space and its interaction with collagen strongly influence the mechanical behavior of the tissue.