In situ analysis of mineral content and crystallinity in bone using infrared micro-spectroscopy of the ν₄ PO₄³⁻ vibration

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 ν₁,ν₃ PO₄³⁻ contour. The ν₄ 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 ν₄ 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 ν₄ PO₄³⁻ FTIR absorption peak ratio of two peaks (603/563 cm⁻¹) within the ν₄ 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 ν₄ PO₄³⁻ contour with the ν₂ CO ₃²⁻ 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 ν₄ 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.

Introduction

Mineral features such as crystallite size and perfection (crystallinity), phosphate content, carbonate content, and mineral environment may be altered substantially as a function of tissue type, age, and pathology. Bone diseases such as osteogenesis imperfecta [1], osteomalacia [2], osteoporosis [3], osteopetrosis [4], and osteoarthritis [5] are characterized by abnormal tissue mineral content, deposition, and/or turnover.

Techniques traditionally used to study bone tissue, namely X-ray diffraction, nuclear magnetic resonance, electron spin resonance, and Fourier transform infrared (FTIR) spectroscopy, while permitting identification of the nature of the mineral phases present, provide neither spatially-resolved analysis of the various mineral phases, nor permit determination of the relative amounts of mineral and matrix present [6]. IR micro-spectroscopy permits chemical analysis of the organic and mineral components of bone [7] such that specific regions of the tissue can be analyzed with spatial resolution approaching the diffraction limit (3–20 μm), making this technique beneficial for understanding mechanisms of abnormal mineral deposition in bone disease.

Following the initial demonstration of the feasibility of the approach [7], IR spectroscopy and micro-spectroscopy of bone have been used extensively to examine protein content [8], [9], phosphate [10], [11], [12], [13] and carbonate [14], [15] content and environment, and mineral crystallinity [16], [17], [18]. Curve-fitting of the ν₁, ν₃ PO₄³⁻ contour (900–1200 cm⁻¹) reveals a variety of components assigned to apatitic phosphate and acid phosphate in environments of varying crystallinity [11]. Subtle changes in this broad contour are detectable as a function of bone crystal maturity [10]. For example, a component at 1145 cm⁻¹, which is assigned to acid phosphate, decreases as bone matures [13]. Also, the 1030/1020 cm⁻¹ intensity ratio, which is an index of crystal size/perfection, increases as bone matures [11]. This parameter has been very useful for recent IR micro-spectroscopic imaging of bone [19], [20].

Acid phosphate content and crystallinity can also be obtained from the ν₄ PO₄³⁻ spectral region (500–650 cm⁻¹). This contour can be fit to fewer components [12] than the ν₁,ν₃ contour, thereby simplifying curve-fitting analysis. However, the ν₄ PO₄³⁻ contour falls in a frequency region below the cutoff of the mercury-cadmium-telluride detectors used in commercial IR microscopes, thereby rendering this vibration generally inaccessible for imaging studies. To circumvent this problem, we recently described the use of a synchrotron infrared microscope coupled to a Cu-doped Germanium detector, which extends the collectable infrared range to 4000–400 cm⁻¹ [21], [22], [23]. Through a 10-μm aperture, the synchrotron infrared source is 1000 times brighter than a conventional globar source. This high brightness permits collection of high quality data with a spatial resolution at the diffraction limit [24]. Since the ν₄ PO₄³⁻ contour is lower in frequency, the diffraction-limited spatial resolution is approximately twice that of the ν₁,ν₃ contour. However, the simplified data analysis and complementary information attainable from this contour make it a valuable region for infrared analysis.

In the present study, synchrotron IR micro-spectroscopy is used to monitor crystallinity and acid phosphate content in bone based on examination of the ν₄ PO₄³⁻ region. To develop spectra–structure correlations, a group of homogenized bone and synthetic hydroxyapatite samples are analyzed using conventional IR spectroscopy. Average crystal size/perfection (crystallinity) is determined from X-ray powder diffraction. These results are correlated with parameters derived from the corresponding IR spectra. Also, acid phosphate content [12] and carbonate content and environment [14] are determined in these samples. This set of trends is then applied to in situ IR micro-spectroscopic analysis of bone samples. The ability to measure relative bone mineral content and composition from within different regions of individual bone samples provides insight into the chemistry of how bone mineral is deposited in a single species.