Preliminary estimates of the calcium/phosphorus ratio at different cortical bone sites using synchrotron microCT.

The Ca/P ratio was measured in cortical bone samples from the femoral neck, front and rear tibia of female rats (1.5 years of age), using synchrotron radiation microtomography. The use of a monoenergetic x-ray beam, as provided by the synchrotron facility, generates accurate 3D maps of the linear attenuation coefficient within the sample and hence gives the ability to map different chemical components. Data sets were taken at 20 keV for each bone sample and calibration phantoms. From the 3D data sets, multiple 2D slices were reconstructed with a slice thickness of approximately 28 microm and converted to Ca/P ratios using the calibration phantom results. Mean values (M +/- SD) for cortical femoral, front and rear tibias are 2.12 +/- 0.08, 1.75 +/- 0.06 and 1.94 +/- 0.07 respectively. These values were compared with those derived from different animals. Differences between the same bone sites from different animals are not significant (0.1 < p < or = 0.9) while those between different bone sites are highly significant (p < 10(-3)) demonstrating a dependence upon life style and bone use.

An automated approach for analyzing D-periods in collagen fibril images.

This paper considers an approach for analyzing fibrillar collagen structures based on fundamental concepts of pattern recognition. It focuses on the quantitative comparison between collagen structural data (electron-optical data) and chemical data. Theoretical models in the form of sequence-generated histograms are used as reference for extracting and analyzing the structural unit in images from collagen fibrils. In this respect, collagen provides a valuable model system for studying the chemical basis of ultrastructure, as well as detecting the alterations in collagen fibril structure produced by a disorder. Application examples are presented and the results are compared with biochemical studies.

Analysis of fibrous proteins from electron microscopy images.

This paper considers an approach for analyzing fibrillar collagen structures from electron microscopy images. It enables the quantitative comparison between collagen structural data (electron-optical data) and chemical data. The particular objectives of the paper are to model the electron microscopy images according to the periodic structure of collagen, provide methods for extracting periodic features directly from the experimental data and propose schemes for comparing these features with the theoretical amino-acid distributions of the examined collagen tissue. Theoretical models in the form of sequence-generated histograms are used as reference for extracting and analyzing the structural unit in images from collagen fibrils. In this respect, collagen provides a valuable model system for studying the chemical basis of ultra-structure and the mechanisms of various treatments on a protein, as well as detecting the alterations in collagen fibril structure produced by a disorder. The algorithms developed in this study can be applied to any fibrous protein, provided that its amino acid sequences and structural properties are known. Several application examples are presented. The algorithmic results are compared with clinical studies as to verify the applicability and potential of the proposed methodology.

High resolution Ca/P maps of bone architecture in 3D synchrotron radiation microtomographic images.

The Ca/P ratio was measured in cortical bone samples from the femoral neck and tibia of different animal species, using synchrotron radiation microtomography. Use of a monoenergetic X-ray beam, as provided by the synchrotron facility, generates accurate 3D maps of the linear attenuation coefficient within the sample and hence gives the ability to map different chemical components. Also, by comparing normal and abnormal bones, i.e. osteoporotic (induced by inflammation), changes in the Ca/P ratio brought about by bone diseases can be detected. MicroCT data sets were collected at 20 and 28 keV for each bone sample and two calibration phantoms. From the 3D data sets, multiple 2D slices were reconstructed with a slice thickness of approximately 30 microm. Regions of interest were defined around suitable sites and were converted to Ca/P ratios using the data collected from the test phantoms. A significant difference (p<0.001) between osteoporotics and age-matched normals at both energies was detected. Differences between different bone sites from the same animal are not significant (p>0.5) while those between the same bone sites from different animals are highly significant (p<0.001). Differences between estimates made at 20 and 28 keV are not significant (p>0.5). An important aspect is the ability to map the spatial distribution of the Ca/P ratio.

Collagen as a model for the study of radiation induced side effects: use of image processing.

Collagen can be used as a model system for studying the effects of radiation on a protein. Quantitative studies of collagen fibrils from electron-optical images from radiated and non-radiated material can produce information not only about the structural effects produced by radiation, but also about the actual part of the fibril being affected.

Structural variations of collagen in normal and pathological tissues: role of electron microscopy.

The spectrum of ultrastructural appearances assumed by collagen in normal and pathological tissues is illustrated using techniques of thin section transmission electron microscopy and computer-assisted analysis. The normal fibrillar collagen types are described in order to provide a basis for comparing other normal and abnormal forms. In normal tissues, the anchoring fibril and basal lamina (basement membrane) represent tissue structures largely containing collagen but differing significantly in organisation from normal types I to III fibrillar collagen. In pathological tissue, deviations from normal fine structure are reflected in abnormal aggregates of collagen fibrils (amianthoid and skeinoid fibres) and abnormalities in fibril diameter and cross-sectional profile. Fibrous and segment long-spacing collagen represent two further organisational variants of collagen, the former found widely in pathological tissues, the latter very rarely. Much remains to be discovered about these abnormal collagen variants-their mode of formation, the cells that produce them, and their roles. They also present a challenge for the collagen biologist formulating hypotheses of collagen fibril assembly and molecular organisation.

Diameter distributions of collagenous tissues in relation to sex. A quantitative ultrastructural study.

A number of factors have been implicated in the regulation of collagen fibril diameter. Electron microscopy analysis was used to investigate the role of sex on fibril diameter. Female mouse skin collagen fibrils' mean diameter values were significantly smaller than those from the male, independent of age. In addition, the female rabbit collagen fibrils had a marked decrease in the mean diameter compared to male, in all the tissues examined (skin, liver, and bone). These data suggest that the collagen fibril diameter is related to sex.

Measurement of the axial periodicity of collagen fibrils using an image processing method.

In this paper, a method is described based on a computer-aided analysis of electron-optical images of collagen fibrils from various tissues, in order to determine the axial periodicity of such fibrils. The method gives information at a level of 2-3nm.

Influence of nutritional factors on bone collagen fibrils in ovariectomized rats.

The influence of both vitamin D(3) and Ca:P ratio on bone collagen fibrils was investigated in ovariectomized rats. Six weeks after ovariectomy the rats were maintained for 80 days with diets containing vitamin D(3) and calcium supplementation. Age-matched ovariectomized animals were fed a normal diet. When vitamin D(3) was increased in the diet, although no effect in fibril organization was observed in relation to that from ovariectomized rats with the normal diet, a highly significant effect in fibril diameter was detected. When the calcium:phosphorus (Ca:P) ratio was increased from 1:1 to 2:1 (without vitamin D(3) supplementation) both structural fiber parameters were significantly affected. The results were closer to normal (i.e., collagen fibrils from animals without ovariectomy) when vitamin D(3) and Ca:P ratios were combined.

Structural alterations in rat skin and bone collagen fibrils induced by ovariectomy.

In this study, the influence of ovariectomy in rat skin and bone (trabecular and cortical) collagen fibrils is examined using electron microscopy. Structural changes (fibril architecture and diameter) were detected, at the ultrastructural level, in skin and bone specimens from ovariectomized rats. The overall collagen fibril architecture was disturbed as compared with normal animals. Treated collagen fibrils' mean diameter values were significantly smaller than those from controls, in all tissues examined. The banding patterns of fibrils were normal in all cases; however, measurements by a computerized method of measuring axial periodicity of fibrils indicated significantly lower values for treated samples than untreated samples. Our results show a correlation between the effects induced by ovariectomy in skin and bone collagen. But, the question of whether these changes play a role in the pathogenesis of ovarian hormone deficiency in osteoporosis remains to be demonstrated.

Image processing of collagen: role in medicine.

This article describes how quantitative studies of electron-optical images from collagen fibrils, used as a model system can provide information not only about the molecular architecture of the fibril but also about the structural alterations produced by a treatment or disorder.

Side effects of lithium treatment on collagenous tissues.

Structural alterations of collagen fibrils induced by treatment with lithium chloride at various doses and durations, are investigated. In addition, this article describes how computer analyses of electron-optical images from collagen fibrils can lead to information about the changes produced by lithium on the molecular architecture of the fibril.

In vivo measurement of radius calcium/phosphorus ratio by X-ray absorptiometry.

We describe a new method for assessing the skeletal Ca/P ratio in vivo using X-ray absorptiometry. By placing cerium (Ce) and samarium (Sm) filters in the X-ray beam from a commercial X-ray source (Norland), mean photon energies of 39 and 89 keV were obtained. The instrument was designed to take measurements of the forearm, at a site located at the distal 1/3 of the radius. The system was calibrated with three bone phantoms: Ca10(PO4)6(OH)2, Ca(HPO4)(H2O)2 and Ca(HPO4)2(H2O)). The precision for measuring the Ca/P ratio in the human radius was 2.3% CV for a skin dose to the forearm ranging from 0.3 to 0.4 mGy, depending on the width of the arm. The Ca/P ratio of the radius was significantly lower in patients with postmenopausal osteoporosis than in premenopausal controls.

The effect of lithium treatment on collagenous tissues: an electron microscope study.

Structural alterations of mouse skin collagen fibrils brought about by treatment with lithium chloride at various doses, as studied by electron microscopy, are discussed. The effect of the duration of treatment on such fibrils is also investigated. An analysis of positive staining patterns from fibrils from mice treated with lithium is included. In addition, this review describes how computer analyses of electron-optical images from collagen fibrils can lead to information about the changes produced by lithium on the molecular architecture of the fibril.

The influence of inflammation-mediated osteopenia (IMO) on the structure of rabbit bone and skin collagen fibrils.

The influence of Inflammation Mediated Osteopenia (IMO) on rabbit skin and bone (trabecular and cortical) collagen fibrils was studied by electron microscopy. Severe abnormalities in collagen fibril structure were detected, at the ultrastructural level, in skin and bone specimens from IMO rabbits. In treated animals the arrangement of fibrils is anarchic. The overall collagen fibril architecture is disturbed compared to normal. IMO collagen fibrils' mean diameter values were significantly larger than those from controls, in all examined tissues. However, the banding patterns of fibrils were normal in all cases. Computer analysis shows no differences in charged amino acid composition between IMO and untreated samples. Our results show a correlation between the effects induced by osteopenia on skin and bone collagen.

The effects of inflammation-mediated osteoporosis (IMO) on the skeletal Ca/P ratio and on the structure of rabbit bone and skin collagen.

The relationships between the skeletal Ca/P ratio (used as an index of bone quality) and structural changes in the bone and skin was studied in inflammatory mediated osteoporosis (IMO). The bone Ca/P ratio in IMO rabbits was significantly lower than in controls. Also, severe alterations were detected at the ultrastructural level in bone and skin collagen fibrils from IMO rabbits.