Measurement of the elastic properties of single osteon lamellae is still one of the most demanding tasks in bone mechanics to be solved. By means of site-matched Raman microspectroscopy, acoustic microscopy and nanoindentation the structure, chemical composition and anisotropic elasticity of individual lamellae in secondary osteons were investigated.

Acoustic impedance images (911-MHz) and two-dimensional Raman spectra were acquired in sections of human femoral bone. The samples were prepared with orientations at various observation angles θ relative to the femoral long axis. Nanoindentations provided local estimations of the elastic modulus and landmarks necessary for spatial fusion of the acoustic and spectral Raman images. Phosphate ν1 (961 cm⁻¹) and amide I (1665 cm⁻¹) band images representing spatial distributions of mineral and collagen were fused with the acoustic images.

Acoustic impedance was correlated with the indentation elastic modulus E_IT (R²=0.61). Both parameters are sensitive to elastic tissue anisotropy. The lowest values were obtained in the direction perpendicular to the femoral long axis. Acoustic images exhibit a characteristic bimodal lamellar pattern of alternating high and low impedance values. Since this undulation was not associated with a variation of the phosphate ν1-band intensity in the Raman images, it was attributed to variations of the lamellar orientation. After threshold segmentation and conversion to elastic modulus the orientation and transverse isotropic elastic constants were derived for individual ensembles of apparent thin and thick lamellae. Our results suggest that this model represents the effective anisotropic properties of an asymmetric twisted plywood structure made of transverse isotropic fibrils.

This is the first report that proves experimentally the ability of acoustic microscopy to map tissue elasticity in two dimensions with micrometer resolution. The combination with Raman microspectroscopy provides a unique way to study bone and mineral metabolism and the relation with mechanical function at the ultrastructural tissue level.