Abstract
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 ν 1,ν 3 PO 4 3− contour. The ν 4 PO 4 3− 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 ν 4 PO 4 3− spectral regions, obtained with a synchrotron radiation source and a Cu-doped Ge detector coupled to an IR microscope. The acid phosphate (HPO 4 2−) 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 ν 4 PO 4 3− FTIR absorption peak ratio of two peaks (603/563 cm −1) within the ν 4 PO 4 3− 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 ν 4 PO 4 3− contour with the ν 2 CO 3 2− 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 PO 4 3− sites in the hydroxyapatite lattice. Thus, IR microscopic analysis of the ν 4 PO 4 3− 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.
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