Applications of Fourier transform infrared microscopy to biomineralization

Poorly crystalline hydroxyapatite (HA) is the major mineral component of calcified tissues. The size and perfection of the crystals vary according to tissue function, age and pathology. X-ray diffraction and transmission electron microscopy are the techniques traditionally used to study this variation, but are limited with regard to either spatial resolution, or adequate identification of the crystal phases. The application of Fourier transform infrared (FT-IR) microscopy to the study of calcified tissues produces information about crystal phases at 20$\mu$ spatial resolution.

To interpret the FT-IR microscopy spectra, it was necessary to examine apatite analogues, and develop a spectroscopic method to assess their crystallinity. Changes in the phosphate $v\sb{1}, v\sb{3}$ region, 900-1200 cm$\sp{-1}$, were shown to reflect changes in the crystallinity of the apatites. The 900-1200 cm$\sp{-1}$ absorbance for a series of synthetic apatites was analyzed by curve fitting techniques and found to be composed of three main sub-bands. The percent area of a component near 1060 cm$\sp{-1}$ decreased as the length of the c-axis of the hydroxyapatite compounds increased, and the position of a band near 1020 cm$\sp{-1}$ increased with increasing crystallinity of the apatites. These spectral signatures are thus proposed as an index of crystallinity of biological hydroxyapatite compounds. FT-IR microscopy was then utilized to study the effects of tissue preservation on the components of bone. Results indicate that fixation of 35 day old intact, and ground rat bone does not significantly alter the crystal size of the apatite. However, formalin-fixation of fetal rat bone altered the structure of the apatite mineral phase. In addition, evaluation of protein secondary structure in 35 day old rat femurs from the Amide I and Amide II vibrations near 1650 and 1550 cm$\sp{-1}$, respectively, revealed that protein conformation was altered by ethanol fixation.

The effects of sodium fluoride therapy on mineral from patients with the degenerative bone disease osteoporosis were then assessed by FT-IR microscopy. An increase in HA crystal size was found after treatment with fluoride in a series of controlled biopsy samples. This work demonstrates the potential of FT-IR microscopy as a diagnostic technique in the field of bone pathology.