Nanoscale modifications in the early heating stages of bone are heterogeneous at the microstructural scale

Nanoscale studies of bone provide key indicators to evidence subtle structural changes that may occur in the biomedical, forensic and archaeological contexts. One specific problem encountered in all those disciplines, for which the identification of nanostructural cues could prove useful, is to properly monitor the effect of heating on bone tissue. In particular, the mechanisms at work at the onset of heating are still relatively unclear. Using a multiscale approach combining Raman microspectroscopy, transmission electron microscopy (TEM), synchrotron quantitative scanning small-angle X-ray scattering imaging (qsSAXSI) and polarized light (PL) microscopy, we investigate the ultrastructure of cortical bovine bone heated at temperatures < 300°C, from the molecular to the macroscopic scale. We show that, despite limited changes in crystal structure, the mineral nanoparticles increase in thickness and become strongly disorganized upon heating. Furthermore, while the nanostructure in distinct anatomical quadrants appears to be statistically different, our results demonstrate this stems from the tissue histology, i.e. from the high degree of heterogeneity of the microstructure induced by the complex cellular processes involved in bone tissue formation. From this study, we conclude that the analysis of bone samples based on the structure and organization of the mineral nanocrystals requires performing measurements at the histological level, which is an advantageous feature of qsSAXSI. This is a critical aspect that extends to a much broader range of questions relating to nanoscale investigations of bone, which could also be extended to other classes of nanostructured heterogeneous materials.