Catherine P. Tarnowski

Three-dimensional cellular development is essential for ex vivo formation of human bone.

Tissue engineering of human bone is a complex process, as the functional development of bone cells requires that regulatory signals be temporally and spatially ordered. The role of three-dimensional cellular interactions is well understood in embryonic osteogenesis, but in vitro correlates are lacking. Here we report that in vitro serum-free transforming growth factor (TGF)-β1 stimulation of osteogenic cells immediately after passage results in the formation of three-dimensional cellular condensations (bone cell spheroids) within 24 to 48 hours. In turn, bone cell spheroid formation results in the up-regulation of several bone-related proteins (e.g., alkaline phosphatase, type I collagen, osteonectin) during days 3–7, and the concomitant formation of micro-crystalline bone. This system of ex vivo bone formation should provide important information on the physiological, biological and molecular basis of osteogenesis.

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.

Earliest Mineral and Matrix Changes in Force‐Induced Musculoskeletal Disease as Revealed by Raman Microspectroscopic Imaging

Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common human birth defect in the skull. Raman microspectroscopy was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force‐induced craniosynostosis. Raman imaging revealed decreased relative mineral content in skulls undergoing craniosynostosis compared with unloaded specimens.

Identification of Outliers in Hyperspectral Raman Image Data by Nearest Neighbor Comparison

A new algorithm for removal of cosmic spikes from hyperspectral Raman image data sets is presented. Spectra in a 3 × 3 pixel neighborhood are used to identify outlier-contaminated data points in the central pixel of that neighborhood. A preliminary despiking of the neighboring spectra is performed by median filtering. Correlations between the central pixel spectrum and its despiked neighbors are calculated, and the most highly correlated spectrum is used to identify outliers. Spike-contaminated data are replaced using results of polynomial interpolation. Because the neighborhood contains spectra obtained in three different frames, even large multi-pixel spikes are identified. Spatial, spectral, and temporal variation in signal is used to accurately identify outliers without the acquisition of any spectra other than those needed to generate the image itself. Sharp boundaries between regions of high chemical contrast do not interfere with outlier identification.

Early mineralization of normal and pathologic calvaria as revealed by Raman spectroscopy

Bone tissue consists of a carbonated apatite-like mineral supported on a hydrated, collagen-rich protein matrix. Despite extensive studies into the macroscopic characteristics of bone, much about the early stages of bone formation remains unknown. Raman microspectroscopy and imaging are increasingly important tools for the study of mineralized tissue, due to advancements in both spectral acquisition and analysis protocols. With this technique, mapping of both organic and inorganic components of bone, in addition to determining their distributions with high spatial resolution across a specimen, can be realized. We have employed Raman microscopy to investigate the early stages of mineralization in four different mouse calvarial systems: typical and atypical osteoblastic (bone forming) cell cultures and healthy and diseased bone tissue. These systems are commonly utilized as models for mineralization. The mineral deposited by osteoblast cultures grown atypically gives a Raman signal completely different to that observed in osteoblast cultures grown in the conventional manner. Similarly, Raman images of healthy and diseased bone tissue show differences in the relationship of the mineral and matrix environments. In this report, we compare the several differences between these four mineral environments, and discuss the chemistry of mineral maturation observed.

Effects of treatment protocols and subcutaneous implantation on bovine pericardium: a Raman spectroscopy study

Using Raman microspectroscopy, we have studied mineral deposition on bovine pericardia, fixed according to three different protocols and either implanted subcutaneously or not implanted (controls). A lightly carbonated apatitic phosphate mineral, similar to that found in bone tissue, was deposited on the surface of a glutaraldehyde-fixed, implanted pericardium. Implanted pericardia fixed in glutaraldehyde followed by treatment in either an 80% ethanol or a 5% octanol/40% ethanol solution did not mineralize on implantation. Collagen secondary structure changes were observed on glutaraldehyde fixation by monitoring the center of gravity of the amide I envelope. It is proposed that the decrease in the amide I center of gravity frequency for the glutaraldehyde-fixed tissue compared to the nonfixed tissue is due to an increase in nonreducible collagen cross-links (1660 cm(-1)) and a decrease in reducible cross-links (1690 cm(-1)). The amide I center of gravity in the glutaraldehyde/ethanol-fixed pericardium was higher than the glutaraldehyde-fixed tissue center of gravity. This increase in center of gravity could possibly be due to a decrease in hydrogen bonding within the collagen fibrils following the ethanol pretreatment. In addition, we found a secondary structure change to the pericardial collagen after implantation: an increase in the frequency of the center of gravity of amide I is indicative of an increase in cross-links.

Raman microscopy of de-novo woven bone tissue

The composition of the bone tissue initially formed during the early mineralization of calvarial bone is poorly understood. Calvarial de novo mineral deposition occurs rapidly; however, whether the mineral is first deposited as an amorphous calcium phosphate or some other calcium phosphate lattice is unclear. Raman microscopy offers the ability to distinguish differences in the mineral lattice through the positions and shifts in the bands of the bone tissue mineral constituents, particularly in the phosphate v1 stretch vibration (950 - 963 cm-1). The ratios of the mineral and organic matrix constituents throughout the sampled region can also elucidate the type of bone tissue deposited. The ability to examine intact specimens at high spatial resolution, without interference from water, is an important feature of Raman microscopy. Using postnatal murine calvaria, we show that the earliest mineral detected is a carbonated hydroxyapatite with other phosphate environments present at lower levels as well. We discuss the mineral composition changes with respect to the age of the mouse over the time period of 2 weeks using a sequence of calvarial sections: postnatal days 3, 7 and 14. We use two different data analysis techniques, factor analysis and center of gravity calculations, to elucidate these discrete changes.

Multivariate data reduction techniques for hyperspectral Raman imaging

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.