Compressive stress on bone, cartilage and other tissues accompanies normal activity. While the biomechanical properties of many tissues are reasonably well-understood at many levels of structure, surprisingly little is known at the ultrastructural and crystal lattice levels. We show how the use of diamond anvil cell Raman microspectroscopy enables a deeper understanding of the response of tissue to mechanical stress. We discuss the reversible responses of deproteinated and intact bone powders to hydrostatic pressure and compare these responses to those of a model compound, synthetic carbonated apatite.
The skeleton is certainly essential to our physiology. Yet, surprisingly little is understood about how bone responds when a load is applied. This is particularly true at the ultrastructural level, where neither the behavior under stress of the collagenous matrix nor that of the calcium phosphate mineral has been explored. Most standard techniques used in the investigation of the biomechanical properties of bone tissue are unable to obtain information at this level. Raman spectroscopy, however, is capable of probing biological specimens at the molecular level, without interference from water or common histological stains. Using this technique, we are able to observe changes in both the mineral and the matrix of bone during and after the application of load. These changes manifest themselves as band shifts in the bone Raman spectrum. We utilize this ability in this study. Two-dimensional Raman imaging is used to investigate the effect of macroscopic fracture on the mineral in sections of femora from 4-month-old and 18-month-old mice. Changes are seen in the mineral around fractured areas that indicate the fracture process may be causing a phase transformation in the bone mineral, similar to the 'transformation toughening' process that is observed in many ceramics. We discuss the physiological implications of these results.
While many macroscopic approaches have been used to study bone's mechanical properties, little is known about the effect of mechanical stresses on bone tissue at the microstructural and submicrostructural levels. Raman microspectroscopy has been found to be an extremely sensitive technique for examining the effects of mechanical loading on polymer structures and thin films, as well as for the study of protein conformations. We present the first application of this technique to bone tissue. The organic component of bone is a highly ordered matrix composed mainly of collagen fibrils. Stress upon the bone tissue creates disorder in this structure; this disorder can easily be detected by Raman spectroscopy. Small changes in the matrix structure manifest themselves as band shifts in the Raman spectra.
Raman microspectroscopy and imaging can be used to probe the chemical properties of newly mineralized bone tissue. In this study, our early mineralization models are neonatal murine cranial suture tissue and prostate cancer cell cultures. The murine cranial tissue was harvested from animals three weeks postnatal. On this time scale, remodeling does not corrupt the temporal record inherent in the spatial distribution of mineral species. When analyzing transects, line images, of the cranial tissue, multivariate data processing is required to generate chemical state plots from the hundreds of Raman spectra acquired during a single transect experiment. In most cranial tissue specimens more than one phosphate mineral environment is observed, allowing inferences on the relation between chemical structure and physiologically important properties. The prostate cancer cell cultures were cultured for up to nine days. Point microspectroscopy reveals the ratios of mineral species present and the amount of protein species in the cell cultures changes dramatically over the course of 9 days. Very low carbonation, typical of early-mineralized tissue, is observed in both of these models.
Underlying the contrast in a hyperspectral Raman image are complete Raman spectra at each of tens or hundreds of thousands of pixels. Multivariate statistics allows reduction of these large data sets to manageable numbers of chemically significant descriptors that become the image contrast. In most cases an object can be viewed as containing a small number (usually fewer than ten) chemically discrete components, each with its own vibrational spectrum. Principal component analysis (PCA) and exploratory factor analysis (FA) can be used to generate descriptors from the experimentally observed Raman spectra in image data sets. Additionally, PCA and FA can be viewed as optimized weighted signal averaging techniques. FA contrast is generated from all regions of a spectrum that are attributable to one component. The result is better signal/noise ratio than is obtained using the height or area of a single band as image contrast. We will discuss a variety of preprocessing steps such as removing outliers and selecting spectral subregions for data analysis optimization. We will illustrate these concepts using an image of bone tissue.
We discuss the use of Raman microprobe spectroscopy and Raman imaging to study the chemical composition of fresh, unmounted bone at a microscopic level. A specimen of human cortical bone was analyzed and evidence for the presence of amorphous-type calcium phosphate, a theoretical precursor in the bone formation process, was found. In general the amorphous4ype calcium phosphate appears away from osteons, in the interstitial tissue. This finding calls into question the role of amorphous-type calcium phosphate as a precursor to apatitic phosphate, since it was not found in the recently remodeled bone near the osteon center, but rather in older bone tissue. Some reasons for the presence of amorphous calcium phosphate are proposed. Possible relations ofthe amorphous mineral to bone damage and bone remodeling are discussed.
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