Richard Mendelsohn

Spectroscopic characterization of collagen cross-links in bone

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross‐linking pattern. The intermolecular cross‐linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross‐linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6‐deficient chick homogenized bone and their age‐ and sex‐matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at ∼1660 cm−1 and ∼1690 cm−1 was related to collagen cross‐links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH‐DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross‐links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (∼5 μm) calcified tissue sections with a spatial resolution of ∼7 μm.

FTIR microscopic imaging of collagen and proteoglycan in bovine cartilage

Articular cartilage, a connective tissue that provides resistance to compressive forces during joint movements, has not been examined in detail by conventional Fourier transform infrared (FTIR) spectroscopy, microspectroscopy (FTIRM), or imaging (FTIRI). The current study reports FTIRM and FTIRI analyses of normal bovine cartilage and identifies the specific molecular components of cartilage that contribute to its IR spectrum. FTIRM data acquired through the superficial, middle, and deep zones of thin sections of bovine articular cartilage showed a variation in intensities of the absorbance bands that arise from the primary nonaqueous components of cartilage, collagen, and proteoglycan (primarily aggrecan) and thus reflected the differences in quantity of these specific components. The spectra of mixtures of model compounds, which had varying proportions of type II collagen and aggrecan, were analyzed to identify spectral markers that could be used to quantitatively analyze these components in cartilage. Collagen and aggrecan were then imaged by FTIRI based on markers found in the model compounds. Polarization experiments were also performed to determine the spatial distribution of the collagen orientation in the different zones of cartilage. This study provides a framework in which complex pathological changes in this heterogeneous tissue can be assessed by IR microscopic imaging.

FTIR microspectroscopic analysis of human osteonal bone

Fourier Transform Infrared Microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of non-osteoporotic human osteonal bone. Spectral maps in four orthogonal directions (in 10 μm steps) from the centers towards the peripheries of individual osteons were obtained from iliac crest biopsies of two necropsy cases. Mineral to matrix ratios, calculated from the ratio of integrated areas of the phosphate v1,v3 band at 900–1200 cm-1 to the amide I band at 1585–1725 cm-1, increased from the center to the periphery of the osteon. The total carbonate (based on the v2 band at ≈850–900 cm-1) to phosphate v1,v3 ratio decreased as the mineral to matrix ratio increased. Analysis of the v2 CO32- band with a combination of second-derivative spectroscopy and curve fitting revealed a decrease in “labile” carbonate, a slight decrease in Type A and a slight increase in Type B carbonate from the center to the periphery of the osteon. Similar analysis of the components of the v1,v3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. These components were assigned by comparison with published frequencies for apatite and acid-phosphate containing calcium phosphates. The most consistent variations were alterations in the relative percent areas of bands at ≈1020 and ≈1030 cm-1, which had previously been assigned to nonstoichiometric and stoichiometric apatites, respectively. This ratio was used as an index of variation in crystal perfection throughout the osteon. This ratio decreased as the mineral to matrix ratio increased. The reproducibility of these parameters at multiple sites in multiple biopsies suggests their applicability for the analysis of mineral changes in disease.

Novel infrared spectroscopic method for the determination of crystallinity of hydroxyapatite minerals

Biologically important apatite analogues have been examined by Fourier Transform Infrared Spectroscopy (FT-IR), and a method developed to quantitatively assess their crystalinity. Changes in the phosphate v1 and v3 regions, 900-1,200 cm-1, for a series of synthetic (containing hydroxide, fluoride, or carbonate ion) and biological apatites with crystal sizes of 100-200 A were analyzed with curve-fitting and second derivative spectroscopy. The v1,v3 contour was composed of three main subbands. Correlations were noted between two spectral parameters and crystal size as determined by x-ray diffraction. The percentage area of a component near 1,060 cm-1 decreased as the length of the c-axis of the hydroxyapatite (HA) compounds increased, while the frequency of a band near 1,020 cm-1 increased with increasing length of the apatite c-axis. These parameters are thus proposed as indices of crystallinity for biological (poorly crystalline) HA. The FT-IR spectra of highly crystalline apatitic compounds were also analyzed. For crystal sizes of 200-450 A, the percentage area of the phosphate v1 band (near 960 cm-1) decreased with increasing HA crystal size. IR indices of crystallinity have thus been developed for both well crystallized and poorly crystallized HA derivatives. The molecular origins of the various contributions to the v1,v3 contour are discussed, and a preliminary application of the method to a microscopic biological sample (rat epiphyseal growth plate) is illustrated.

Fourier transform infrared spectroscopy of the solution-mediated conversion of amorphous calcium phosphate to hydroxyapatite: New correlations between X-ray diffraction and infrared data

Fourier Transform infrared spectroscopic analysis of maturing, poorly crystalline hydroxyapatite (HA) formed from the conversion of amorphous calcium phosphate (ACP) at constant pH or variable pH show only subtle changes in the ν1, ν3 phosphate absorption region (900 cm−1−1200 cm−1). This region is of interest because it can ve detected by analysis of mineralized tissue sections using FT-IR microscopy. To evaluate the subtle spectral changes occurring during the maturation, second derivatives of the spectra were calculated. HA formed at constant pH showed little or no variation in the second derivative peak positions with bands occurring at 960 cm−1, 985 cm−1, 1030 cm−1, 1055 cm−1, 1075 cm−1, 1096 cm−1, 1116 cm−1, and 1145 cm−1. These bands can be assigned to molecular vibrations of the phosphate (PO43−) moiety in an apatitic/stoichiometric environment of HA. In contrast, during the early stages of maturation of the HA formed at variable pH, second derivative peak positions occurring at 958 cm−1, 985 cm−1, 1020 cm−1, 1038 cm−1, 1112 cm−1, and 1127 cm−1 shifted in position with maturation, indicating, that the environment of the phosphate species is changing as the crystals mature. Peaks at 1020 cm−1, 1038 cm−1, 1112 cm−1, and 1127 cm−1 were attributable to nonstoichiometry and/or the presence of acid phosphate-containing species. This concept was supported by the lower Ca:P molar ratios measured by chemical analysis of the synthetic material made at variable pH. Using the second derivative peak positions as initial input parameters, the ν1, ν3 phosphate region of the synthetic HAs prepared at constant pH were curve fit. X-ray diffraction patterns of these same materials were also curve fit to calculate the changes in crystallinty (size/perfection) in the c-axis 002 reflection as well as the 102, 210, 211, 112, 300, 200, and 301 planes. Linear regression analysis showed that the changes in the percent area of the underlying bands at 982 cm−1, 999 cm−1, 1030 cm−1, 1075 cm−1, 1096 cm−1, 1116 cm−1, and 1145 cm−1 were correlated with changes in crystallinity in one or more of the reflection planes. It is suggested that a combination of second-derivative and curve-fitting analysis of the ν1, ν3 phosphate contour allows the most reproducible evaluation of these spectra.

External Infrared Reflection Absorption Spectrometry of Monolayer Films at the Air-Water Interface

The theory and practice of external infrared reflection absorption spectrometry (IRRAS) as applied to monomolecular films at the air-water interface are reviewed. The observed IR frequencies for films of amphiphilic species provide information about the conformational states of the hydrocarbon chains and the hydrogen bonding and ionization states of the polar head groups, under conditions of controlled surface pressure. Determination of molecular orientation is also feasible and requires detailed consideration of the reflection-absorption properties of the three- phase (air-monolayer-water) system. Current theoretical approaches are described. Applications of IRRAS to the study of single- and double-chain amphiphiles and proteins are reviewed, and initial excursions into biochemistry (interfacial enzyme catalysis) and physiology (pulmonary surfactant function) are reported.

In situ analysis of mineral content and crystallinity in bone using infrared micro-spectroscopy of the ν4 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 nu(1),nu(3) PO(4)(3-) contour. The nu(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 nu(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 nu(4) PO(4)(3-) FTIR absorption peak ratio of two peaks (603/563 cm(-1)) within the nu(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 nu(4) PO(4)(3-) contour with the nu(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 nu(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.

Complementary information on bone ultrastructure from scanning small angle X-ray scattering and Fourier-transform infrared microspectroscopy

Scanning small angle X-ray scattering (scanning SAXS) and Fourier-transform infrared microspectroscopy (FT-IRM) have previously been utilized independently to characterize the structural properties of bone in an anatomical position-resolved fashion. Whereas SAXS provides a direct measure of the physical characteristics of apatitic crystals, FT-IRM assesses structure of both mineral and organic matrix at the molecular level. In the present study both methods were applied to examine the same developing bone tissue from the L-4 vertebra of a 14-month-old (accidental death). A 200-microm-thick section was processed for examination by scanning electron microscopy and SAXS. Spectra were collected at 200 microm spatial resolution at specific locations in cortical and cancellous bone. Parameters determined included total SAXS intensity, crystal thickness (T), and degree and direction of predominant crystal orientation. For FT-IRM analysis, a section 4 microm thick was cut longitudinally from the top of the sample. Spectra of regions 100 x 100 microm2 were acquired from the same locations as the SAXS spectra. Integrated areas of the phosphate nu(1,3) collagen amide I, and carbonate nu2 absorbances, were calculated to obtain mineral: matrix and carbonate:mineral ratios. The relative quantities of types A, B, and labile carbonate (substituted for apatite hydroxyl, phosphate, and surface positions, respectively) were also evaluated. Polarized FT-IRM data were collected to determine molecular orientation of the apatite and collagen components. The results of this study show that the information obtained from the two techniques is complementary. Both SAXS and FT-IRM data revealed that the crystals were significantly larger in the cancellous region compared with the cortical region, that mineralization was greater in the cortex, and that the crystals were oriented to a larger degree in the cancellous compared with the cortical bone. The scanning SAXS measure of crystal thickness was significantly correlated to the FT-IRM measures of crystallinity, type A carbonate substitution, and crystal orientation. In conclusion, it was found that the combined use of SAXS and FT-IRM provides valuable, unique information on structural changes in bone at both the microstructural and ultrastructural level. Although each method can be used individually, the combination of techniques provides additional insights into the mechanism of bone crystal maturation.

Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation

Fourier-transform infrared microspectroscopy (FTIRM) allows analysis of mineral content, mineral crystal maturity and mineral composition at ~10-μ spatial resolution. Previous FTIRM analyses comparing 4-μ thick sections from non-decalcified iliac crest biopsies from women with post-menopausal osteoporosis, as contrasted with iliac crest tissue from individuals without evidence of metabolic bone disease, demonstrated significant differences in average mineral content (decreased in osteoporosis) and mineral crystal size/perfection (increased in osteoporosis). More importantly, these parameters, which vary throughout the tissue in relation to the tissue age in healthy bone, showed no such variation in bone biopsies from patients with osteoporosis. The present study compares the spatial and temporal variation in mineral quantity and properties in trabecular bone in high- and low-turnover osteoporosis. Specifically, six biopsies from women (n=5) and one man with high-turnover osteoporosis (age range 39–77) and four women and two men with low turnover osteoporosis (age range 37–63) were compared to ten “normal” biopsies from three men and seven woman (age range: 27–69). “High turnover” was defined as the presence of increased resorptive surface, higher than normal numbers of osteoclasts and greater than or equal to normal osteoblastic activity. “Low turnover” was defined as lower than normal resorptive surface, decreased osteoclast number and less than normal osteoblastic activity. Comparing variations in FTIR-derived values for each of the parameters measured at the surfaces of the trabecular bone to the maximum value observed in multiple trabeculae from each person, the high-turnover samples showed little change in the mineral: matrix ratio, carbonate: amide I ratio, crystallinity and acid phosphate content. The low-turnover samples also showed little change in these parameters, but in contrast to the high-turnover samples, the low-turnover samples showed a slight increase in these parameters, indicative of retarded, but existent resorption and formation. These data indicate that FTIR microspectroscopy can provide quantitative information on mineral changes in osteoporosis that are consistent with proposed mechanisms of bone loss.

Vibrational spectroscopic studies of lipid domains in biomembranes and model systems

The Singer–Nicolson fluid mosaic model of membrane structure (Singer and Nicolson, 1972) depicting the lipid bilayer as a fluid, homogeneous solvent for membrane proteins has been augmented in recent years to include domain formation as a major driving force in membrane organization (Metcalfe et al., 1986; Welti and Glaser, 1994). Domains may be functionally significant as regulators of membrane protein activity and as a means of arranging cell surface receptors. Many plasma membranes contain morphologically non-uniform regions such as synapses, gap junctions, microvilli, etc. The basolateral and apical domains of polarized epithelial cells have different protein and lipid compositions, with the domains separated by gap junctions. Evidence is also accumulating for heterogeneous lipid distributions in membranes without specialized functions (Kinnunen, 1991; Rodgers and Glaser, 1993; Yang and Glaser, 1995). Even in simple systems such as phospholipid mixtures in their La (liquid crystalline) phases, organized spatial distributions and superlattice arrangements have been observed (Cheng et al., 1997; Liu et al., 1997). Several terms exist in the literature to describe non-random distributions of lipid molecules, e.g. domain formation, microaggregation, clustering, phase separation, microphage separation or lateral phase separation. The reader is cautioned that no consensus exists as to which term is appropriate for a particular morphology. The most straightforward non-random distribution arises from phospholipid gel state immiscibility in a binary mixture, where spatially distinct regions may be temporally quite stable. Such structures are generally deduced from experimentally determined phase diagrams. When the gel phase of a lipid mixture is warmed, gel and fluid lipid phases simultaneously coexist. At the microscopic level, lipid spatial organization may vary from random mixtures to a very non-ideal distribution of constituent phases. In systems containing at least some conformational order, Mouritsen and coworkers have used Monte Carlo simulations to examine both time-dependent and time-independent clustering of phospholipids in two-component mixtures (Jorgensen et al., 1993; Jorgensen and Mouritsen, 1995; Mouritsen, 1995), while * Corresponding author. Tel.: +1 201 6485329; fax: +1 201 6481264.