Mary M. J. Tecklenburg

Carbonate Assignment and Calibration in the Raman Spectrum of Apatite

A series of apatites with varying carbonate levels was prepared in order to assign the carbonate bands and calibrate for Raman analysis of natural materials. Overlap of carbonate bands with phosphate peaks was resolved by curve fitting. A peak at 1,071 cm−1 was assigned to a combination of the carbonate ν1 mode at 1,070 cm−1 with a phosphate ν3 mode at 1,076 cm−1. In addition, the carbonate ν4 mode was identified in apatite samples with >4% carbonate. The carbonate ν4 bands at 715 and 689 cm−1 identify the samples as B-type carbonated apatite. The carbonate content of apatite was calibrated to a carbonate Raman band, and the method was used to determine the carbonate content of a sample of bovine cortical bone, 7.7 ± 0.4%.

Highly ordered interstitial water observed in bone by nuclear magnetic resonance.

NMR was used to study the nanostructure of bone tissue. Distance measurements show that the first water layer at the surface of the mineral in cortical bone is structured. This water may serve to couple the mineral to the organic matrix and may play a role in deformation.

Bone Chemical Structure Response to Mechanical Stress Studied by High Pressure Raman Spectroscopy

While the biomechanical properties of bone are reasonably well understood at many levels of structural hierarchy, surprisingly little is known about the response of bone to loading at the ultrastructural and crystal lattice levels. In this study, our aim was to examine the response (i.e., rate of change of the vibrational frequency of mineral and matrix bands as a function of applied pressure) of murine cortical bone subjected to hydrostatic compression. We determined the relative response during loading and unloading of mineral vs. matrix, and within the mineral, phosphate vs. carbonate, as well as proteinated vs. deproteinated bone. For all mineral species, shifts to higher wave numbers were observed as pressure increased. However, the change in vibrational frequency with pressure for the more rigid carbonate was less than for phosphate, and caused primarily by movement of ions within the unit cell. Deformation of phosphate on the other hand, results from both ionic movement as well as distortion. Changes in vibrational frequencies of organic species with pressure are greater than for mineral species, and are consistent with changes in protein secondary structures such as alterations in interfibril cross-links and helix pitch. Changes in vibrational frequency with pressure are similar between loading and unloading, implying reversibility, as a result of the inability to permanently move water out of the lattice. The use of high pressure Raman microspectroscopy enables a deeper understanding of the response of tissue to mechanical stress and demonstrates that individual mineral and matrix constituents respond differently to pressure.

Natural-Abundance 43Ca Solid-State NMR Spectroscopy of Bone

Structural information about the coordination environment of calcium present in bone is highly valuable in understanding the role of calcium in bone formation, biomineralization, and bone diseases like osteoporosis. While a high-resolution structural study on bone has been considered to be extremely challenging, NMR studies on model compounds and bone minerals have provided valuable insight into the structure of bone. Particularly, the recent demonstration of (43)Ca solid-state NMR experiments on model compounds is an important advance in this field. However, application of (43)Ca NMR is hampered due to the low natural-abundance and poor sensitivity of (43)Ca. In this study, we report the first demonstration of natural-abundance (43)Ca magic angle spinning (MAS) NMR experiments on bone, using powdered bovine cortical bone samples. (43)Ca NMR spectra of bovine cortical bone are analyzed by comparing to the natural-abundance (43)Ca NMR spectra of model compounds including hydroxyapatite and carbonated apatite. While (43)Ca NMR spectra of hydroxyapatite and carbonated apatite are very similar, they significantly differ from those of cortical bone. Raman spectroscopy shows that the calcium environment in bone is more similar to carbonated apatite than hydroxyapatite. A close analysis of (43)Ca NMR spectra reveals that the chemical shift frequencies of cortical bone and 10% carbonated apatite are similar but the quadrupole coupling constant of cortical bone is larger than that measured for model compounds. In addition, our results suggest that an increase in the carbonate concentration decreases the observed (43)Ca chemical shift frequency. A comparison of experimentally obtained (43)Ca MAS spectra with simulations reveal a 3:4 mol ratio of Ca-I/Ca-II sites in carbonated apatite and a 2.3:3 mol ratio for hydroxyapatite. 2D triple-quantum (43)Ca MAS experiments performed on a mixture of carbonated apatite and the bone protein osteocalcin reveal the presence of protein-bound and free calcium sites, which is in agreement with a model developed from X-ray crystal structure of the protein.

Crystallinity and compositional changes in carbonated apatites: Evidence from 31P solid-state NMR, Raman, and AFM analysis

Solid-state (magic-angle spinning) NMR spectroscopy is a useful tool for obtaining structural information on bone organic and mineral components and synthetic model minerals at the atomic-level. Raman and 31P NMR spectral parameters were investigated in a series of synthetic B-type carbonated apatites (CAps). Inverse 31P NMR linewidth and inverse Raman PO43- ν1 bandwidth were both correlated with powder XRD c-axis crystallinity over the 0.3-10.3 wt% CO32- range investigated. Comparison with bone powder crystallinities showed agreement with values predicted by NMR and Raman calibration curves. Carbonate content was divided into two domains by the 31P NMR chemical shift frequency and the Raman phosphate ν1 band position. These parameters remain stable except for an abrupt transition at 6.5 wt% carbonate, a composition which corresponds to an average of one carbonate per unit cell. This near-binary distribution of spectroscopic properties was also found in AFM-measured particle sizes and Ca/P molar ratios by elemental analysis. We propose that this transition differentiates between two charge-balancing ion-loss mechanisms as measured by Ca/P ratios. These results define a criterion for spectroscopic characterization of B-type carbonate substitution in apatitic minerals.

Vibrational characterization of azobenzenes, azoxybenzenes and azoaromatic and azoxyaromatic polyethers

Raman and infrared spectra of a bisphenol-A-containing azoaromatic polyether, a bisphenol-A-containing azoxyaromatic polyether and a bisphenol-A-containing 2-hydroxyazoaromatic polyether were analyzed. Vibrational spectra were also collected for the corresponding model compounds 4,4′-(4-tert-butylphenoxy)azobenzene, 4,4′-(4-tert-butylphenoxy)azoxybenzene and 4,4′-(4-tert-butylphenoxy)-2-hydroxyazobenzene. Comparisons with substituted azobenzenes and azoxybenzenes were used in assigning the spectra in the 1650–900 cm-1 region. Assignments of the N=N stretch (1410–1390 cm-1) demonstrate that all of the compounds are in the trans configuration. The azo bridge vibrations of the azoaromatic ethers are very similar to those of azobenzene. Different types of hydrogen bonding are seen in the 2-hydroxyazoaromatic compounds. The polyether has an OH stretch at 3355 cm-1 which indicates a network of intermolecular hydrogen bonds among the polymers. The 2-hydroxyazoaromatic model compound has no IR peak above 3100 cm-1 and N=N, CO and CN stretches which are shifted due to an intramolecular hydrogen bond with the azo nitrogen. The N=N stretch of the azoxyaromatic ethers is at the same wavenumber as in the azo compounds. Assignment of the N→O stretch vibration is difficult because of mixing with other modes of the azoxy bridge.

Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: An ultrastructural, compositional and comparative analysis with mouse bone

Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone.

Noninvasive Raman spectroscopy of rat tibiae: approach to in vivo assessment of bone quality.

Abstract. We report on in vivo noninvasive Raman spectroscopy of rat tibiae using robust fiber-optic Raman probes and holders designed for transcutaneous Raman measurements in small animals. The configuration allows placement of multiple fibers around a rat leg, maintaining contact with the skin. Bone Raman data are presented for three regions of the rat tibia diaphysis with different thicknesses of overlying soft tissue. The ability to perform in vivo noninvasive Raman measurement and evaluation of subtle changes in bone composition is demonstrated with rat leg phantoms in which the tibia has carbonated hydroxylapatite, with different carbonate contents. Our data provide proof of the principle that small changes in bone composition can be monitored through soft tissue at anatomical sites of interest in biomedical studies.

Green synthesis of gold nanoparticles reduced and stabilized by squaric acid and supported on cellulose fibers for the catalytic reduction of 4-nitrophenol in water

We report a simple, fast, and green method to produce gold nanoparticles (AuNPs) that are reduced and stabilized by sodium squarate in water and easily attach to cellulose fibers. The AuNPs and its nanocomposites with cellulose fibers exhibited excellent catalytic activity for the reduction of 4-nitrophenol (4-NP) with NaBH4. A glass column was packed with the nanocomposites and used for the continuous catalytic reduction of 4-NP with NaBH4 and demonstrated to be used multiple times (20×) without loss of catalytic activity.

Effect of copper on the local structure of GeSe2Cux probed by Raman spectroscopy

Glassy GeSe2 is known to exhibit photodarkening that is extinguished by the presence of copper. Bulk glasses of GeSe2Cux (0<x<0.16), prepared by melt-quenching, were studied by Raman spectroscopy to ascertain the effect of copper on the local structure of the glass. Decreases were observed in relative areas of the A1c (218 cm−1) and Ge–Ge (178 cm−1) peaks relative to the A1 (201 cm−1) for compositions of less than x=0.05 copper but changed very little at higher compositions. The position of the A1 and A1c peaks decreased by 2 cm−1 in low copper compositions. The density of the samples also increased sharply below x=0.05 and more gradually above x=0.05. The copper is seen as affecting the defects (Ge–Ge bonds) in the stoichiometric germanium–selenium glass but not affecting the tetrahedral Ge(Se1/2)4 chains.