Brief communication: Two and three-dimensional analysis of bone mass and microstructure in a bog body from the Iron Age

Human remains from peat bogs, called "bog bodies," have yielded valuable insights into human history because of their excellent preservation of soft tissue. On the other hand, the acidic environment of the peat leads to an extensive demineralization of skeletal elements, complicating their analysis. We studied the skeleton of the bog body "Moora" dated to approximately 650 B.C. Nondestructive evaluation of the bone was made using contact X-rays, peripheral quantitative computed tomography (pQCT) analysis, multislice computed tomography (CT) and high resolution micro computed tomography (microCT) imaging. Two thousand seven hundred years in the acidic environment of the bog led to a loss of 92.7% of bone mineral density. Despite this demineralization and in contrast to other bog bodies, the spatial structure of the bones of "Moora" is exceptionally well preserved. We found Harris lines and were able to obtain the first three-dimensional data on the trabecular microstructure of the bone of a young woman from the early Iron Age.     

Diagnostic value of micro-CT in comparison with histology in the qualitative assessment of historical human postcranial bone pathologies.

Micro-computed tomography (muCT) is of great interest for palaeopathological examination because it is less invasive than histology. This study evaluates the diagnostic value of muCT for postcranial macerated bones. We investigated five specimens (osteomyelitis, tuberculosis, trauma, osteosarcoma and hypertrophic osteoarthropathy) of a pathology reference series by muCT and polarised light microscopy. The 3D muCT images allow an easy orientation within the specimen. Surface structures, thickness, continuity of the cortex and number, thickness and orientation of the trabeculae can be evaluated. The high number of muCT slices helps to choose the most interesting areas for further investigations. Grey value images display the degree of mineralisation. Yet, the differentiation between woven and lamellar bone is only possible using polarised light microscopy. muCT is a tool of high value for the examination of postcranial bone disorders. It cannot replace histological examinations completely because it cannot assess the bone quality (woven or lamellar). For the choice of the optimal location where slices for the microscopic investigation are later cut in heterogeneous samples, muCT is very useful. Therefore, we suggest performing the muCT examination first, followed by histology if necessary.     

Analysis of biomechanical effects on bone and on the muscle-bone interactions in small animal models

Animal models are suitable to study many aspects of bone structure and strength. This article reviews some general principles of current bone biomechanics and describes the scope of the available methodology for biomechanical studies of the musculoskeletal system employing those models. The analysis comprises bone and muscle "mass" indicators provided by standard densitometry (DEXA); bone 'mass', 'apparent density', geometry or architectural design and strength and muscle strength indicators that can be determined by peripheral quantitative computed tomography (pQCT), and bone material and structural (whole-bone) properties than can be directly assessed by destructive mechanical tests. Some novel interrelationships that can be investigated that way are discussed, namely, 1. the pathogenetic analysis of the effects on whole-bone strength, 2. the discrimination between mineralization and microstructural factors as determinants of changes in the bone material or structural properties, 3. the evaluation of the interaction of a treatment with the ability of bone 'mechanostat' to optimize the bone architectural design by 'distribution / mass' and 'distribution / quality' curves, and 4. the analysis of effects on the musclebone interactions for a differential diagnosis between 'physiological' or 'disuse' and 'true' osteopenias and osteoporoses.     

Assessing the accuracy of high-resolution X-ray computed tomography of primate trabecular bone by comparisons with histological sections

Different lines of evidence suggest that trabecular bone architecture contains a functional signal related to an organism's locomotor behavior. An understanding of the interspecific and intraspecific variation in extant nonhuman primate trabecular structure is needed to evaluate its usefulness as a tool to reconstruct the locomotor habits of extinct primates. High-resolution X-ray computed tomography (HRXCT) is a new imaging approach with a resolution in the tens of microns that allows nondestructive access to the internal structure of bony elements. Previous studies indicate that such resolution is necessary to accurately quantify structural parameters of trabecular bone. The primary goal of this study was to test the accuracy of HRXCT by comparing stereological measurements from HRXCT images and histological thin sections of cancellous bone taken from the proximal femur and humerus of baboons. To this end, 11 bone samples were scanned on an HRXCT scanner and then thin-sectioned to reveal the scanned plane. HRXCT images were thresholded using a modified half-maximum height protocol. The stereological measurements included bone volume fraction (BV/TV), trabecular number (Tb.N), bone surface to volume ratio (BS/BV), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). The measurement errors on the HRXCT images were 10.90% for BV/TV, 6.06% for Tb.N, 14.19% for BS/BV, 14.33% for Tb.Th, and 7.09% for Tb.Sp, but none of these measurements were significantly different from the histological standards (alpha = 0.05). A second goal of this study was to examine the influence of thresholding, a necessary step in any morphometric study using computed tomography, on the accuracy of the quantitative morphometry. Threshold values derived from a modified half-maximum height protocol showed that parameters derived from the region of interest (area in which stereological measurements were later taken) produced better reconstructions of the actual bone structure than threshold values derived from more inclusive areas of bone. We conclude that HRXCT can accurately reconstruct the complex architecture of trabecular bone, and that thresholding is a nontrivial step in trabecular bone studies, with even slight changes in the protocol greatly affecting the morphometric data. HRXCT represents a valuable analytical tool that should be of interest to a great many researchers in physical anthropology because it allows nondestructive access to internal morphology, thereby preserving valuable and limited skeletal collections.     

Anatomy of the Cranial Endocast of the Bottlenose Dolphin, Tursiops truncatus, Based on HRXCT

Endocranial surfaces, volumes, and interconnectivities of extant and fossil odontocetes potentially offer information on the general architecture of the brain and on the structure of the specialized cetacean circulatory system. Although conventional methods for acquiring such data have generally involved invasive preparation of the specimen, particularly in the case of fossils, new tomographic technologies afford nondestructive access to these internal morphologies. In this study we used high-resolution X-ray computed tomography (HRXCT) to scan a skull of the extant Tursiops truncatus (Cetacea: Odontoceti). We processed the data to reveal the cranial endocast and details of internal skeletal architecture (data at www.digimorph.org). Major features that can be discerned include aspects of the specimen's hypertrophied retia mirabilia, the major canals and openings of the cranial cavity, and the relationship of the brain and endocranial circulatory structures to the surrounding skeleton. CT data also provide information on the shape of the brain that may be lost in conventional anatomical preparations, and readily provide volumetric and linear measurements of the endocast and its individual segments. These results demonstrate the utility of HRXCT for interpreting the internal cranial anatomy of both extant and fossil cetaceans.     

Application of electron tomography to fungal ultrastructure studies

Access to structural information at the nanoscale enables fundamental insights into many complex biological systems. The development of the transmission electron microscope (TEM) has vastly increased our understanding of multiple biological systems. However, when attempting to visualize and understand the organizational and functional complexities that are typical of cells and tissues, the standard 2-D analyses that TEM affords often fall short. In recent years, high-resolution electron tomography methods, coupled with advances in specimen preparation and instrumentation and computational speed, have resulted in a revolution in the biological sciences. Electron tomography is analogous to medical computerized axial tomography (CAT-scan imaging) except at a far finer scale. It utilizes the TEM to assemble multiple projections of an object which are then combined for 3-D analyses. For biological specimens, tomography enables the highest 3-D resolution (5 nm spatial resolution) of internal structures in relatively thick slices of material (0.2-0.4 microm) without requiring the collection and alignment of large numbers of thin serial sections. Thus accurate and revealing 3-D reconstructions of complex cytoplasmic entities and architecture can be obtained. Electron tomography is now being applied to a variety of biological questions with great success. This review gives a brief introduction into cryopreservation and electron tomography relative to aspects of cytoplasmic organization in the hyphal tip of Aspergillus nidulans.     

Treatment of osteoporosis with human parathyroid peptide and observations on effect of sodium fluoride.

OBJECTIVE--To evaluate the need for a randomised study of treatment of spinal osteoporosis with human parathyroid peptide in the secondary prevention of crush fractures; to study the effect of human parathyroid hormone peptide 1-34 plus sex hormones on vertebral body cancellous bone; and, separately, to determine the effect of relatively low doses of sodium fluoride plus calcium on spinal bone mineral density. DESIGN--Open study of patients with primary or postmenopausal osteoporosis. All patients had serial bone densitometry of the spine by quantitative computed tomography and dual photon absorptiometry as well as serial densitometry of the radial midshaft (cortical) and radial distal (trabecular) bone by quantitative computed tomography. Changes in the spinal bone not forming the spongiosa of the vertebral bodies ("cortical" bone) were determined from the difference between the two axial measurements, after correction to the same units of measurement. SETTING--Northwick Park Hospital and Medical Research Council Clinical Research Centre. PATIENTS--24 Patients who fulfilled the conventional criteria for type 1 (vertebral) osteoporosis not secondary to recognised causes other than sex hormone deficiency and with at least one crush or wedge vertebral fracture and a spinal bone density (quantitative computed tomography) less than 80 mg/cm3 or two or more fractures. Twelve patients received human parathyroid peptide and 12 sodium fluoride; they were not randomised. MAIN OUTCOME MEASURES--Trends in axial and peripheral bone mass values determined by linear, time dependent regression analyses. RESULTS--The patients receiving the peptide showed a substantial increase in vertebral spongiosa (mean 25.6 mg/cm2 two years after the start of treatment). No significant changes were seen in spinal cortical or radial bone density. The patients receiving sodium fluoride showed roughly equal increases in cancellous and cortical bone over the same period (mean increase in vertebral spongiosa 16.1 mg/cm3). No significant changes were seen in radial bone. CONCLUSIONS--Treatment of postmenopausal women with human parathyroid peptide selectively increases spinal cancellous bone density by amounts that may prove useful in secondary prevention. Peptide treatment should now be tested in a randomised study in which the important end point is prevention of fractures as the usefulness of sodium fluoride in this context is doubtful.     

Densitometric, morphometric and mechanical distributions in the human proximal femur

With the prevalent use of DXA-measured BMD to assess pathologic hip fractures and its recently reported lack of reliability to predict fracture or account for efficacy of anti-resorptive therapy, it is reasonable to assess whether variations in the primary and secondary tensile and compressive trabecular microstructure can account for variations in proximal femur strength in comparison to DXA-measured BMD. To that end, microstructural and densitometric measures of trabecular bone specimens, from discrete sites within the proximal femur, were correlated with their mechanical properties. We hypothesize that accounting for regional variations in trabecular microstructure will improve predictions of proximal femur strength and stiffness compared to bone density measured by DXA. Forty-seven samples (seven donors) from seven distinct sites of human proximal femur underwent DXA and muCT imaging and mechanical testing. The results revealed significant variations in BMC, morphometric indices and mechanical properties within the proximal femur. This work has demonstrated that the mechanical performance of each sub-region is highly dependent on the corresponding trabecular microstructure. BMD measured by DXA at standard regions of interest cannot resolve the variations in trabecular density and microstructure that govern the mechanical behavior of the proximal femur. This work suggests that a quantitative Singh index that uses high resolution QCT to monitor the trabecular microstructure at specific sub-regions of the proximal femur may allow better predictions of hip fracture risk in individual patients and an improved assessment of changing bone structure in response to pharmacological interventions.     

Peripheral quantitative computed tomography of the distal radius in young subjects - new reference data and interpretation of results

Application of bone densitometry to clinical use requires the availability of accessible reference data and is helped by an interpretative framework that is based on bone physiology. The aim of this contribution is to provide both reference data and help in the interpretation of results for peripheral quantitative computed tomography (Stratec XCT2000 performed at the distal radius of young subjects. Data from a previous reference data study on 478 subjects between 6 and 40 years were re-analyzed and smooth curves were fitted. The corresponding equations allow for calculation of age- and sex-specific z-scores of bone mineral content, volumetric bone mineral density (vBMD) of the trabecular compartment, vBMD of the entire radial cross-section, total cross-sectional area and cortical thickness. These data should facilitate the clinical use of peripheral quantitative computed tomography in young subjects.     

Increased bone mass in male and female mice following tamoxifen administration

Tamoxifen is capable of preserving bone mass in gonadectomized rodents as well as intact female mice; however, a detailed 3D quantitative analysis of the structural changes produced in the growing skeleton of intact mice of both genders by this agent is lacking. Employing quantitative microcomputed tomography (muCT), we assessed the effects of 4-hydroxytamoxifen (OHT) on the femora of C57BL/6J mice administered this agent either for 12 (males and females) or 2 (females) weeks. In mice of either gender, but especially in females, 12 weeks of OHT exposure led to dramatic increases in both cortical and trabecular bone. Females exposed to OHT for either 2 or 12 weeks demonstrated significantly increased cortical wall thickness, trabecular bone volume, connectivity, and number, as well as decreased trabecular separation. Significant increases in several of these parameters were also evident in males after 12 weeks of OHT administration. In view of the expanding use of OHT to induce Cre-mediated recombination events, our findings suggest that care should be exercised when interpreting the skeletal phenotypes of mice exposed this agent, particularly in situations where the effects of OHT might synergize with the phenotypic outcome of a specific genetic alteration.     

Automated most-probable loss tomography of thick selectively stained biological specimens with quantitative measurement of resolution improvement

We describe the technique and application of energy filtering, automated most-probable loss (MPL) tomography to intermediate voltage electron microscopy (IVEM). We show that for thick, selectively stained biological specimens, this method produces a dramatic increase in resolution of the projections and the computed volumes versus standard unfiltered transmission electron microscopy (TEM) methods. This improvement in resolution is attributed to the reduction of chromatic aberration, which results from the large percentage of inelastic electron-scattering events for thick specimens. These improvements are particularly evident at the large tilt angles required to improve tomographic resolution in the z-direction. This method effectively increases the usable thickness of selectively stained samples that can be imaged at a given accelerating voltage by dramatically improving resolution versus unfiltered TEM and increasing signal-to-noise versus zero-loss imaging, thereby expanding the utility of the IVEM to deliver information from within specimens up to 3 microm thick.     

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear‐staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro‐CT) imaging is a versatile, well‐documented tool traditionally used to generate high‐resolution 3‐D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro‐CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro‐CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro‐CT measurement of fetal limb lengths correlates well with traditional clear‐staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro‐CT and 10.82% for clear‐staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro‐CT technique to generate 3‐D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear‐staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well. Birth Defects Res (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

Monochromatic synchrotron radiation muCT reveals disuse-mediated canal network rarefaction in cortical bone of growing rat tibiae.

The purpose of this study was to demonstrate the ability of computed microtomography based on monochromatic synchrotron radiation (SRmuCT) in microstructural analysis of cortical bone. Tibial diaphyses of growing rats (14 wk, n = 8) undergoing unilateral sciatic neurectomy 8 wk before study were imaged with spatial volume resolution of 5.83 x 5.83 x 5.83 microm3 by SRmuCT (20 keV) at the synchrotron radiation facility (SPring-8). Reconstructed image data were translated into local mineral densities by using a calibrated linear relationship between linear absorption coefficients and concentrations of homogeneous K2HPO4 solution. Pure bone three-dimensional images, produced by simple thresholding at a bone mineral density of 0.82 g/cm3, were analyzed for macro- and microscopic structural properties. In neurectomized hindlimbs, cortical canal network rarefaction as well as bone atrophy were found. The former was characterized by 30% smaller porosity, 11% smaller canal density in transverse section, and 38% smaller canal connectivity density than those in contralateral bone. On the other hand, no difference was found in bone mineral density between neurectomized and intact hindlimbs (1.37 vs. 1.36 g/cm3). In conclusion, SRmuCT is a promising method for the three-dimensional analysis of cortical microstructure and the degree of mineralization in small animals.     

Imaging of angiogenesis: from microscope to clinic

Advances in imaging are transforming our understanding of angiogenesis and the evaluation of drugs that stimulate or inhibit angiogenesis in preclinical models and human disease. Vascular imaging makes it possible to quantify the number and spacing of blood vessels, measure blood flow and vascular permeability, and analyze cellular and molecular abnormalities in blood vessel walls. Microscopic methods ranging from fluorescence, confocal and multiphoton microscopy to electron microscopic imaging are particularly useful for elucidating structural and functional abnormalities of angiogenic blood vessels. Magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), ultrasonography and optical imaging provide noninvasive, functionally relevant images of angiogenesis in animals and humans. An ongoing dilemma is, however, that microscopic methods provide their highest resolution on preserved tissue specimens, whereas clinical methods give images of living tissues deep within the body but at much lower resolution and specificity and generally cannot resolve vessels of the microcirculation. Future challenges include developing new imaging methods that can bridge this resolution gap and specifically identify angiogenic vessels. Another goal is to determine which microscopic techniques are the best benchmarks for interpreting clinical images. The importance of angiogenesis in cancer, chronic inflammatory diseases, age-related macular degeneration and reversal of ischemic heart and limb disease provides incentive for meeting these challenges.     

Skeletal deformations in medaka (Oryzias latipes) visualized by synchrotron radiation micro-computer tomography (SRmicroCT)

Synchrotron radiation micro-computer tomography (SRmicroCT) offers the possibility to investigate biomineralized structures in high detail. Two animals of adult medaka fish (Oryzias latipes) were analyzed by SRmicroCT with a resolution of 6.55 microm: the wild-type animal was normally developed whereas the second animal showed an idiopathic deformation of the cranial and axial skeleton. These deformations could be followed on the macro- and on the microscale (i.e., on the level of the individual ribs and fin bones). Our study clearly demonstrates that SRmicroCT is an excellent technique to study alterations in the skeletal structure of fish in detail.     

Comparison of radiometric and histometric features of bone samples from ribs

OBJECTIVE: To correlate quantitative data gathered from conventional radiograms as well as from quantitative computed tomography (QCT) images of bone samples with histometric coefficients of the same specimens. STUDY DESIGN: Bone samples were obtained from standardized sites of ribs during autopsy of 40 males aged 15-84 years. In the bone samples the following assessments were performed: densitometry of bone using QCT, estimation of radiostructural quantitative coefficients (coefficient of trabecular arrangement anisotropy, trabecular thickness coefficient); histometry (area of bone matrix, trabecular thickness coefficient); relative weights of dry, decalcified bone and bone in ash form; and concentration of calcium in ash. Radiometry and histometry were performed using image analysis methods with IBAS for Windows (Kontron, Eching b. München, Germany) equipment. Radiostructure of bone was evaluated with the use of our own algorithm; concentration of calcium was measured spectrophotometrically. Analysis of variance, cross correlation, regression analysis and others were the statistical tools applied. RESULTS: From the variables analyzed, the following indicates a significant correlation with the histometric area of bone matrix: radiologic trabecular anisotropy, QCT densitometry (especially of cancellous bone) and the ratio of weight of dry sample to fresh sample. The coefficient of radiologic trabecular thickness appeared not to be a valuable feature for estimation of bone histology. CONCLUSION: Quantitative estimation of radiostructure of bone carried out on conventional radiograms may be helpful in the detection of histologic properties of bone, an important factor in assessment of the fragility of bone.     

生物组织的超声调制光学成像技术

具有超声定位的高空间分辨率和光学检测的高灵敏度的超声调制光学成像技术是一种有前途的无损的生物组织成像技术。文章阐述了该技术的成像原理,评述了前人在散射介质中声光作用机制的理论研究;介绍了该领域在技术路线上的最新研究进展;最后总结了超声调制光学成像技术的优点并展望了其在生物医学领域的应用前景。     

光声成像及其在生物医学中的应用

光声成像是一种新近迅速发展起来、基于生物组织内部光学吸收差异、以超声作媒介的无损生物光子成像方法,它结合了纯光学成像的高对比度特性和纯超声成像的高穿透深度特性的优点,以超声探测器探测光声波代替光学成像中的光子检测,从原理上避开了光学散射的影响,可以提供高对比度和高分辨率的组织影像,为研究生物组织的结构形态、生理特征、代谢功能、病理特征等提供了重要手段,在生物医学临床诊断以及在体组织结构和功能成像领域具有广泛的应用前景.对光声成像技术的机理、光声成像技术和方法、光声图像重建算法以及光声成像在生物医学上的应用情况作一个简单介绍,希望有助于推动我国在该领域的科研和开发应用工作的迅速发展.     

Utility of high resolution x-ray computed tomography (HRXCT) for paleobotanical studies: an example using London Clay fruits and seeds

High resolution x-ray computed tomography (HRXCT) was used to image pyritized fossil fruits from the Lower Eocene London Clay flora to test the utility of this technique for paleobotanical application. The combination of carbon-pyrite preservation and void spaces between fruit and seed layers within fossils provides differences in density and composition that enable excellent imaging. Fossil fruits of Palaeorhodomyrtus subangulata (Bowerbank) Reid & Chandler (Myrtaceae) were investigated in situ within their silicone fluid conservation medium, which protects these unstable fossils from oxygen and humidity. HRXCT recovers taxonomically informative anatomical and morphological detail and provides a means of nondestructive examination of delicate type materials and other important specimens. These results suggest that HRXCT will be applicable to a broad spectrum of pyritized fossils to record structural details in inherently unstable materials.