Study of composition and structure of demineralized bone using X-ray techniques

The aim of this study is to compare the composition and structure of cortical bones with different stages of in-vitro demineralization from the micro (10–500 μm) to the nano-scale level (below 1 μm) using X-ray techniques, such as Small Angle X-ray Scattering (SAXS), X-ray Microfluorescence (μ-XRF) and Synchrotron Microtomography (SR-μCT). In total, 8 fresh cortical bone samples of different ages from 2 diaphyseal femora were selected. They were submitted to a 4 days demineralization experiment with 2 concentrations of EDTA acid (0.1 mol L−1 pH 10, and 0.5 mol L−1 pH 7.4, at 25 °C, without agitation). EDTA is a slow demineralization agent and permits better tissue preservation of bone specimens. μ-XRF elemental mapping showed that EDTA 0.1 mol L−1 demineralized more homogeneously the samples than EDTA 0.5 mol.L−1 (pH 7.4). SR-μCT data have demonstrated the changes that occurred in the 3D internal microstructure of cortical bone during the demineralization and besides the results concluded that both EDTA concentrations had similar effect on the intracortical porosity. From SAXS analysis EDTA 0.1 mol L−1 had a better performance in the demineralization of the samples without damaging the nanostructures.