In situ analysis of mineral content and crystallinity in bone using infrared micro-spectroscopy of the v4 PO43- vibration

Measurements of bone mineral content and composition in situ provide insight into the chemistry of bone mineral deposition. Infrared (IR) micro-spectroscopy is well suited for this purpose. To date, IR microscopic (including imaging) analyses of bone apatite have centered on the v(1),v(3) PO43- contour. The v(4) PO43- contour (500-650 cm(-1)), which has been extensively used to monitor the crystallinity of hydroxyapatite in homogenized bone samples, falls in a Frequency region below the cutoff of the mercury-cadmium-telluride detectors used in commercial IR microscopes, thereby rendering this vibration inaccessible For imaging studies. The current study reports the first IR micro-spectroscopy spectra of human iliac crest cross sections in the v(4) PO43- spectral regions. obtained with a synchrotron radiation source and a Cu-doped Ge detector coupled to an IR microscope. The acid phosphate (HPO42-) content and mineral crystallite perfection (crystallinity) of a human osteon were mapped. To develop spectra-structure correlations. a combination of X-ray powder diffraction data and conventional Fourier transform IR spectra have been obtained from a series of synthetic hydroxyapatite crystals and natural bone powders of various species and ages. X-ray powder diffraction data demonstrate that there is an increase in average crystal size as bone matures, which correlates with an Increase In the v(4) PO43- FTIR absorption peak ratio of two peaks (603/563 cm(-1)) within the v(4) PO43- contour. Additionally, the IR results reveal that a band near 540 cm(-1) may be assigned to acid phosphate. This band is present at high concentrations in new bone, and decreases as bone matures. Correlation of the v(4) PO43- contour with the v(2) CO32- contour also reveals that when acid phosphate content is high. type A carbonate content (i.e.. carbonate occupying OH- sites in the hydroxyapatite lattice) is high. As crystallinity increases and acid phosphate content decreases, carbonate substitution shifts toward occupation of PO43- sites in the hydroxyapatite lattice. Thus. IR microscopic analysis of the v(4) PO43- contour provides a straightforward index of both relative mineral crystallinity and acid phosphate concentration that can be applied to in situ IR micro-spectroscopic analysis of bone samples. which are of interest for understanding the chemical mechanisms of bone deposition in normal and pathological states. (C) 2001 Elsevier Science B.V, All rights reserved.