Renal osteodystrophy alters metabolic activity and remodeling rate of bone and also may lead to different bone composition. The objective of this study was to characterize the composition of bone in high-turnover renal osteodystrophy patients by means of Fourier transform infrared spectroscopic imaging (FTIRI). Iliac crest biopsies from healthy bone (n = 11) and patients with renal osteodystrophy (ROD, n = 11) were used in this study. The ROD samples were from patients with hyperparathyroid disease. By using FTIRI, phosphate-to-amide I ratio (mineral-to-matrix ratio), carbonate-to-phosphate ratio, and carbonate-to-amide I ratio (turnover rate/remodeling activity), as well as the collagen cross-link ratio (collagen maturity), were quantified. Histomorphometric analyses were conducted for comparison. The ROD samples showed significantly lower carbonate-to-phosphate (p < .01) and carbonate-to-amide I (p < .001) ratios. The spatial variation across the trabeculae highlighted a significantly lower degree of mineralization (p < .05) at the edges of the trabeculae in the ROD samples than in normal bone. Statistically significant linear correlations were found between histomorphometric parameters related to bone-remodeling activity and number of bone cells and FTIRI-calculated parameters based on carbonate-to-phosphate and carbonate-to-amide I ratios. Hence the results suggested that FTIRI parameters related to carbonate may be indicative of turnover and remodeling rate of bone. © 2010 American Society for Bone and Mineral Research

The macro- and micro-features of bone can be assessed by using imaging methods. However, nano- and molecular features require more detailed characterization, such as use of e.g., vibrational spectroscopy and X-ray scattering. Nano- and molecular features also affect the mechanical competence of bone tissue. The aim of the present study was to reveal the effects of mineralization and its alterations on the mineral crystal scale, by investigating the spatial variation of molecular composition and mineral crystal structure across the cross-section of femur diaphyses in young rats, and healthy and osteoporotic mature rats (N=5). Fourier transform infrared spectroscopy and scanning small- and wide-angle X-ray scattering (SAXS/WAXS) techniques with high spatial resolution were used at identical locations over the whole cross-section. This allowed quantification of point-by-point information about the spatial distribution of mineral crystal volume. All measured parameters (crystal dimensions, degree of orientation and predominant orientation) varied across the cortex. Specifically, the crystal dimensions were lower in the central cortex than in the endosteal and periosteal regions. Mineral crystal orientation followed the cortical circumference in the periosteal and endosteal regions, but was less well-oriented in the central regions. Central cortex is formed rapidly during development through endochondral ossification. Since rats possess no osteonal remodeling, this bone remains (until old age). Significant linear correlations were observed between the dimensional and organizational parameters, e.g., between crystal length and degree of orientation (R(2)=0.83, p<0.001). Application of SAXS/WAXS provides valuable information on bone nanostructure and its constituents, effects of diseases and, prospectively, mechanical competence.

The molecular composition of the organic and inorganic matrices of bone undergoes alterations during maturation. The aim of this study was to compare Fourier transform infrared (FT-IR) and near-infrared (NIR) Raman microspectroscopy techniques for characterization of the composition of growing and developing bone from young to skeletally mature rabbits. Moreover, the specificity and differences of the techniques for determining bone composition were clarified. The humeri of female New Zealand White rabbits, with age range from young to skeletally mature animals (four age groups, n = 7 per group), were studied. Spectral peak areas, intensities, and ratios related to organic and inorganic matrices of bone were analyzed and compared between the age groups and between FT-IR and Raman microspectroscopic techniques. Specifically, the degree of mineralization, type-B carbonate substitution, crystallinity of hydroxyapatite (HA), mineral content, and collagen maturity were examined. Significant changes during maturation were observed in various compositional parameters with one or both techniques. Overall, the compositional parameters calculated from the Raman spectra correlated with analogous parameters calculated from the IR spectra. Collagen cross-linking (XLR), as determined through peak fitting and directly from the IR spectra, were highly correlated. The mineral/matrix ratio in the Raman spectra was evaluated with multiple different peaks representing the organic matrix. The results showed high correlation with each other. After comparison with the bone mineral density (BMD) values from micro-computed tomography (micro-CT) imaging measurements and crystal size from XRD measurements, it is suggested that Raman microspectroscopy is more sensitive than FT-IR microspectroscopy for the inorganic matrix of the bone. In the literature, similar spectroscopic parameters obtained with FT-IR and NIR Raman microspectroscopic techniques are often compared. According to the present results, however, caution is required when performing this kind of comparison.

Collagen is the most prominent protein in biological tissues. Tissue fixation is often required for preservation or sectioning of the tissue. This may affect collagen nanostructure and potentially provide incorrect information when analyzed after fixation. We aimed to unravel the effect of 1) ethanol and formalin fixation and 2) 24h air-dehydration on the organization and structure of collagen fibers at the nano-scale using small and wide angle X-ray scattering. Samples were divided into 4 groups: ethanol fixed, formalin fixed, and two untreated sample groups. Samples were allowed to air-dehydrate in handmade Kapton pockets during the measurements (24h) except for one untreated group. Ethanol fixation affected the collagen organization and nanostructure substantially and during 24h of dehydration dramatic changes were evident. Formalin fixation had minor effects on the collagen organization but after 12h of air-dehydration the spatial variation increased substantially, not evident in the untreated samples. Generally, collagen shrinkage and loss of alignment was evident in all samples during 24h of dehydration but the changes were subtle in all groups except the ethanol fixed samples. This study shows that tissue fixation needs to be chosen carefully in order to preserve the features of interest in the tissue.