Michael D. Morris

Application of vibrational spectroscopy to the study of mineralized tissues (review)

The infrared and Raman spectroscopy of bone and teeth tissues are reviewed. Characteristic spectra are obtained for both the mineral and protein components of these tissues. Vibrational spectroscopy is used to study the mineralization process, to define the chemical structure changes accompanying bone diseases, and to characterize interactions between prosthetic implants and tissues. Microspectroscopy allows acquisition of spatially resolved spectra, with micron scale resolution. Recently developed imaging modalities allow tissue imaging with chemical composition contrast.

Subsurface probing in diffusely scattering media using spatially offset Raman spectroscopy

We describe a simple methodology for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The technique is based on the collection of Raman scattered light from surface regions that are laterally offset away from the excitation laser spot on the sample. The Raman spectra obtained in this way exhibit a variation in relative spectral intensities of the surface and subsurface layers of the sample being investigated. The data set is processed using a multivariate data analysis to yield pure Raman spectra of the individual sample layers, providing a method for the effective elimination of surface Raman scatter. The methodology is applicable to the retrieval of pure Raman spectra from depths well in excess of those accessible with conventional confocal microscopy. In this first feasibility study we have differentiated between surface and subsurface Raman signals within a diffusely scattering sample composed of two layers: trans-stilbene powder beneath a 1 mm thick over-layer of PMMA (poly(methyl methacrylate)) powder. The improvement in contrast of the subsurface trans-stilbene layer without numerical processing was 19 times. The potential applications include biomedical subsurface probing of specific tissues through different overlying tissues such as assessment of bone quality through skin, providing an effective noninvasive means of screening for bone degeneration, other skeletal disease diagnosis, and dermatology studies, as well as materials and catalyst research.

Raman Assessment of Bone Quality

BackgroundProgress in the diagnosis and prediction of fragility fractures depends on improvements to the understating of the compositional contributors of bone quality to mechanical competence. Raman spectroscopy has been used to evaluate alterations to bone composition associated with aging, disease, or injury.Questions/purposesIn this survey we will (1) review the use of Raman-based compositional measures of bone quality, including mineral-to-matrix ratio, carbonate-to-phosphate ratio, collagen quality, and crystallinity; (2) review literature correlating Raman spectra with biomechanical and other physiochemical measurements and with bone health; and (3) discuss prospects for ex vivo and in vivo human subject measurements.MethodsISI Web of Science was searched for references to bone Raman spectroscopy in peer-reviewed journals. Papers from other topics have been excluded from this review, including those on pharmaceutical topics, dental tissue, tissue engineering, stem cells, and implant integration.ResultsRaman spectra have been reported for human and animal bone as a function of age, biomechanical status, pathology, and other quality parameters. Current literature supports the use of mineral-to-matrix ratio, carbonate-to-phosphate ratio, and mineral crystallinity as measures of bone quality. Discrepancies between reports arise from the use of band intensity ratios rather than true composition ratios, primarily as a result of differing collagen band selections.ConclusionsRaman spectroscopy shows promise for evaluating the compositional contributors of bone quality in ex vivo specimens, although further validation is still needed. Methodology for noninvasive in vivo assessments is still under development.

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%.

Ultrastructural changes accompanying the mechanical deformation of bone tissue: a Raman imaging study.

Raman spectroscopy and imaging are known to be valuable tools for the analysis of bone, the determination of protein secondary structure, and the study of the composition of crystalline materials. We have utilized all of these attributes to examine how mechanical loading and the resulting deformation affects bone ultrastructure, addressing the hypothesis that bone spectra are altered, in both the organic and inorganic regions, in response to mechanical loading/deformation. Using a cylindrical indenter, we have permanently deformed bovine cortical bone specimens and investigated the ultrastructure in and around the deformed areas using hyperspectral Raman imaging coupled with multivariate analysis techniques. Indent morphology was further examined using scanning electron microscopy. Raman images taken at the edge of the indents show increases in the low-frequency component of the amide III band and high-frequency component of the amide I band. These changes are indicative of the rupture of collagen crosslinks due to shear forces exerted by the indenter passing through the bone. However, within the indent itself no evidence was seen of crosslink rupture, indicating that only compression of the organic matrix takes place in this region. We also present evidence of what is possibly a pressure-induced structural transformation occurring in the bone mineral within the indents, as indicated by the appearance of additional mineral factors in Raman image data from indented areas. These results give new insight into the mechanisms and causes of bone failure at the ultrastructural level.

Mineralization of Developing Mouse Calvaria as Revealed by Raman Microspectroscopy

Raman microspectroscopy is a nondestructive vibrational spectroscopic technique that permits the study of organic and mineral species at micron resolution, offers the ability to work with hydrated and dehydrated specimens in vivo or in vitro, and requires minimal specimen preparation. We used Raman microspectroscopy to determine the composition of the mineral environments present in mouse calvaria, the flat bones that comprise the top of the skull. We have acquired Raman transects (lines of point spectra) from mouse calvaria during a developmental time course ranging from embryonic day 13.5 (E13.5; 6 days before birth) to 6 months of age. Exploratory factor analysis (FA) reveals the presence of a variety of apatitic mineral environments throughout the tissue series. The earliest mineral is observed in the fetal day 15.5 (F15.5) mice and is identified as a carbonated apatite. The presence of a heterogeneous mineralized tissue in the postnatal specimens suggests that ionic incorporation and crystal perfection in the lattice vary as the mouse develops. This variation is indicative of the presence of both recently deposited mineral and more matured remodeled mineral. Band area ratios reveal that the mineral/matrix ratio initially increases, reaches a plateau, and then increases again. The carbonate/phosphate band area ratio remains constant from F18.5 to postnatal day 3 (PN3) and then increases with age. Insights into the chemical species, the degree of mineralization, and the multiple mineral environments that are present in normal calvarial tissue will enable us to better understand both normal and abnormal mineralization processes.

Numerical Simulations of Subsurface Probing in Diffusely Scattering Media Using Spatially Offset Raman Spectroscopy

We present the first elementary model predicting how Raman intensities vary for a range of experimental variables for spatially offset Raman spectroscopy (SORS), a recently proposed technique for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The model was able to reproduce the key observations made from the first SORS experiments, namely the dependence of Raman signal intensities on the spatial offset between the illumination and collection points and the relative contributions to the overall spectrum from the top layer and sub-layer. The application of the SORS concept to a three-layer system is also discussed. The model also clearly indicates that an annular geometry, rather than a point-collection geometry, which was used in the earlier experiments, would yield much improved data.

Transcutaneous fiber optic Raman spectroscopy of bone using annular illumination and a circular array of collection fibers

Transcutaneous bone Raman spectroscopy with an exciting annulus of 785-nm laser light surrounding the field of view of a circular array of collection fibers is demonstrated. The configuration provides distributed laser light. The annulus is located 2 to 3 mm beyond the edge of the field of view of the collection fibers to reject contributions from skin and other overlying tissues. Data are presented for rat and chicken tissue. For rat tibia, the carbonate/phosphate ratio measured at a depth of 1 mm below the skin is in error by 2.3% at an integration time of 120 s and within 10% at a 30-s integration time. For chicken tibia 4 mm below the skin surface, the error is less than 8% with a 120-s integration time.

Brittle IV mouse model for osteogenesis imperfecta IV demonstrates postpubertal adaptations to improve whole bone strength.

The Brtl mouse model for type IV osteogenesis imperfecta improves its whole bone strength and stiffness between 2 and 6 months of age. This adaptation is accomplished without a corresponding improvement in geometric resistance to bending, suggesting an improvement in matrix material properties.

Chemical Microstructure of Cortical Bone Probed by Raman Transects

Raman transects, microspectra taken at equal intervals along a line, are used to explore the microstructure of human cortical bone. Transects of 50 spectra taken at 2.5 μm intervals across an osteon show spatial differences in local mineral and protein composition as different physiological structures are traversed. Differences in mineral composition are seen near the rim of an osteon and further out in the lamellae. The blood vessel wall, primarily composed of collagen and elastin, is detected inside the Haversian canal. Factor analysis is used to explore the data set and reveals differences in mineral composition. Factor analysis also yields one bone matrix component, an osteoidal tissue component, and one blood vessel protein component. The 4 cm−1 spectral resolution and 2.5 μm spatial sampling facilitate the development of univariate metrics for bone development and health. Band integration is performed for important marker bands including phosphate v1 at 960 cm−1, monohydrogen phosphate v1 at 1003 cm−1, B-type carbonate v1 at ∼ 1070 cm−1, collagen CH2 wag at ∼ 1450 cm−1, and collagen amide I at ∼ 1650 cm−1. Mineral-to-matrix ratio, phosphate-to-monohydrogen phosphate ratio, and carbonate-to-phosphate ratio are calculated from these measured areas.