Feasibility of detecting trabecular bone around percutaneous titanium implants in rabbits by in vivo microfocus computed tomography

Objectives: The goal of this study was to examine the feasibility of in vivo imaging of trabecular bone around titanium implants by means of microfocus computed tomography (micro‐CT) and the use of rabbits for this purpose. Materials and Methods: Ten male rabbits type Hollander, received a titanium implant (1.7 mm diameter and 10 mm length) in the trabecular bone of the left tibia. Seven weeks later a micro‐CT scan was taken. Four rabbits were used to monitor potential harmful effects from X‐ray absorption until 4 weeks after scanning. A second group of six rabbits was used for testing the hypothesis that a good correlation exists between in vivo micro‐CT images and histological images of trabecular bone around titanium implants. The six rabbits were scanned and sacrificed immediately. The tibias were extracted and submitted to standard histological procedures. This resulted in a total of 12 histological sections and their corresponding 12 micro‐CT images. Bone area measurements were performed at the left and right side of the implant in three regions: 0–500, 500–1000 and 1000–1500 μm distance from the implant interface. Intra‐class correlations (ICC) were calculated between both techniques. Results: The four rabbits did not show any sign of radiodermatitis 4 weeks after scanning. In the micro‐CT images of the group of six rabbits, trabeculae are visible, but not well defined, due to the presence of noise in the image. The ICC for the right implant side were 0.44 for zone 0–500 μm, 0.48 for zone 500–1000 μm and 0.40 for zone 1000–1500 μm. The ICC for the left implant side could not be calculated. Conclusion: A low agreement was found between the bone measurements from histology and in vivo micro‐CT images. The use of the in vivo micro‐CT for trabecular bone imaging around metallic implants should be restricted to track tendencies in follow‐up studies.     

Methodological principles for fractal analysis of trabecular bone

A standardized methodology for the fractal analysis of histological sections of trabecular bone has been established.
A modified box counting method has been developed for use on a PC-based image analyser. The effect of image analyser settings, magnification, image orientation and threshold levels was determined. Also, the range of scale over which trabecular bone is effectively fractal was determined and a method formulated to calculate objectively more than one fractal dimension from the modified Richardson plot.
The results show that magnification, image orientation and threshold settings have little effect on the estimate of fractal dimension. Trabecular bone has a lower limit below which it is not fractal (λ < 25 μm) and the upper limit is 4250 μm. There are three distinct fractal dimensions for trabecular bone (sectional fractals), with magnitudes greater than 1.0 and less than 2.0.
It has been shown that trabecular bone is effectively fractal over a defined range of scale. Also, within this range, there is more than one fractal dimension, describing spatial structural entities. Fractal analysis is a model-independent method for describing a complex multifaceted structure, which can be adapted for the study of other biological systems. This may be at the cell, tissue or organ level and complements conventional histomorphometric and stereological techniques.     

A biomechanical study of osteoporotic vertebral trabecular bone: The use of micro-CT and high-resolution finite element analysis

Osteoporosis is one of the most dangerous skeletal diseases in relation to the highest fracture risk in vertebral bones. A considerable amount of work has been done to investigate the biomechanical characteristics of osteoporotic vertebral trabecular bone. Previous researchers studied the elastic characteristics using a micro-finite element (micro-FE) model, used to analyze realistic trabecular architectures in full detail, based on micro-computed tomography (μCT). Since osteoporotic compression fracture is closely associated with the mechanical characteristics of the vertebral trabecular bone and there were few micro-FE models to account for all of the elastic and plastic characteristics in vertebral trabecular bone, this study analyzed the effect of voxel resolution on the plastic characteristics as well as the elastic characteristics of three-dimensional (3D) osteoporotic lumbar trabecular bone models. Also, we evaluated the effect of specimen geometry on this problem. It has been reported that a cubic specimen with side length 6.5mm was suggested as standard specimens for the experimental test of trabecular bone. Current study examined whether or not the effect of the specimen geometry on the experimental test may be also applied to the simulated compression test of trabecular bone specimens. The experimental test employing the rapid prototyping (RP) technique and INSTRON test machine is performed to indirectly validate the results of the simulated compression test by micro-FE analysis. The review finished with the verification about the effects of the simulated compression test.     

基于自回归倒谱法估计骨小梁间距

基于超声背散射信号，本文提出用自回归（AR）倒谱法来估计松质骨中骨小梁的间距。对牛胫骨和人离体跟骨松质骨中的超声背散射信号采用AR倒谱法进行了分析，并估计了牛胫骨和人离体跟骨中骨小梁的平均间距。结果表明，松质骨的密度与骨小梁的平均间距具有密切的关系，随松质骨表观密度的减小，骨小梁的平均间距增大，用AR倒谱能比较准确地估计出松质骨中骨小梁的平均间距。     

A new method for three-dimensional skeleton graph analysis of porous media: application to trabecular bone microarchitecture

This paper introduces a new three-dimensional analysis of complex disordered porous media. Skeleton graph analysis is described and applied to trabecular bone images obtained by high resolution magnetic resonance imaging. This technique was developed bearing in mind topological considerations. The correspondence between vertices and branches of the skeleton graph and trabeculae is used in order to get local information on trabecular bone microarchitecture. In addition to real topological parameters, local structural information about trabeculae, such as length and volume distributions, are obtained. This method is applied to two sets of samples: six osteoporosis and six osteoarthritis bone samples. We demonstrate that skeleton graph analysis is a powerful technique to describe trabecular bone microarchitecture.     

股骨颈密质骨和松质骨抗弯变形的测量方法与初步分析

提出一种新的方法,对股骨颈部位总体、密质骨和松质骨的承载特点进行分析,为今后探求这些特点与股骨颈骨折愈合关系提供基础。通过粘贴应变片和将股骨颈剖开,分别测量不同载荷下股骨颈在保持和去除松质骨时的弯曲变形。结果证明,载荷与应变成线性关系;股骨颈处有、无松质骨在同样载荷和弯曲方向时的应变之比在50％～70％之间。尽管股骨颈部位由松质骨和密质骨组成,且几何形状不规则,受力复杂,但通过粘贴应变仍可测量出股骨颈总体、密质骨和松质骨各自的承载特点：股骨颈处密质骨和松质骨总体为线弹性性质;股骨颈处密质骨的承载能力大于松质骨0～20％。     

Estimation of structural anisotropy based on volume orientation. A new concept

The quantification of anisotropy—its main direction and the degree of dispersion around it—is desirable in numerous research fields dealing with physical structures. Conventional methods are based on the orientation of interface elements. The results of these methods do not always agree with perceived anisotropy, and anisotropic structures do not necessarily turn out to be ‘anisotropic’ using these methods. In the present paper, we propose an alternative to curve and surface orientation, namely volume orientation. Using trabecular bone as an example of a two-phase anisotropic structure, the new concept is studied in some detail. In particular, a parametric method of estimating volume orientation from sections is presented and discussed.     

Estimating length density and quantifying anisotropy in skeletal muscle capillaries

The accurate estimation of stereological parameters defined on anisotropic structures is a long-standing problem. In this paper we seek to estimate the capillary length density J_v in skeletal muscle tissue. A well-known model for directional anisotropy in space, namely the ‘spherical normal’ or ‘Fisher axial distribution’ model, is found to fit the relevant data satisfactorily. Based on this model, a short-cut estimation method is proposed and illustrated with a numerical example. This method essentially consists in taking the ratio of mean capillary profile counts, as obtained from transversal and longitudinal sections of the muscle tissue, and making use of a table or a graph given in the paper to estimate J_v. The conditions under which the methods are applicable and practicable are discussed in detail. Apart from an accurate estimation of J_v, an important feature of our method is the possibility of quantifying the degree of anisotropy by a coefficient K (called the concentration parameter of the Fisher axial distribution), which enjoys both a biological significance and a sound statistical basis.     

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 μm Araldite-embeddedhistologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 μm Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and “expansion chambers” may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.     

Influence of the induced anisotropy in powder-based materials

Development of anisotropy, induced during processes of compaction of metal powders and plastic deformation of sintered metals is considered. A mechanical model, taking into account the induced anisotropy is proposed. The application of the finite element (FE) method for the proposed model is given and a FE code is created. Numerical simulation of compaction and of forging processes are given, showing the influence of the induced anisotropy on the relative density distribution and on the form of the end product.     

Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies

Stereology applied on histological sections is the ‘gold standard’ for obtaining quantitative information on cancellous bone structure. Recent advances in micro computed tomography (µCT) have made it possible to acquire three-dimensional (3D) data non-destructively. However, before the 3D methods can be used as a substitute for the current ‘gold standard’ they have to be verified against the existing standard. The aim of this study was to compare bone structural measures obtained from 3D µCT data sets with those obtained by stereology performed on conventional histological sections using human tibial bone biopsies. Furthermore, this study forms the first step in introducing the proximal tibia as a potential bone examination location by peripheral quantitative CT and CT. Twenty-nine trabecular bone biopsies were obtained from autopsy material at the medial side of the proximal tibial metaphysis. The biopsies were embedded in methylmetacrylate before µCT scanning in a Scanco µCT 40 scanner at a resolution of 20 × 20 × 20 µm³, and the 3D data sets were analysed with a computer program. After µCT scanning, 16 sections were cut from the central 2 mm of each biopsy and analysed with a computerized method. Trabecular bone volume (BV/TV) and connectivity density (CD) were estimated in both modalities, whereas trabecular bone pattern factor (TBPf) was estimated on the histological sections only. Trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp), and structure model index (SMI) were estimated with the µCT method only. Excellent correlations were found between the two techniques for BV/TV (r = 0.95) and CD (r = 0.95). Additionally, an excellent relationship (r = 0.95) was ascertained between TBPf and SMI. The study revealed high correlations between measures of bone structure obtained from conventional 2D sections and 3D µCT data. This indicates that 3D µCT data sets can be used as a substitute for conventional histological sections for bone structural evaluations.     

Three-dimensional surface texture visualization of bone tissue through epifluorescence-based serial block face imaging

Serial block face imaging is a microscopy technique in which the top of a specimen is cut or ground away and a mosaic of images is collected of the newly revealed cross-section. Images collected from each slice are then digitally stacked to achieve 3D images. The development of fully automated image acquisition devices has made serial block face imaging more attractive by greatly reducing labour requirements. The technique is particularly attractive for studies of biological activity within cancellous bone as it has the capability of achieving direct, automated measures of biological and morphological traits and their associations with one another. When used with fluorescence microscopy, serial block face imaging has the potential to achieve 3D images of tissue as well as fluorescent markers of biological activity. Epifluorescence-based serial block face imaging presents a number of unique challenges for visualizing bone specimens due to noise generated by sub-surface signal and local variations in tissue autofluorescence. Here we present techniques for processing serial block face images of trabecular bone using a combination of non-uniform illumination correction, precise tiling of the mosaic in each cross-section, cross-section alignment for vertical stacking, removal of sub-surface signal and segmentation. The resulting techniques allow examination of bone surface texture that will enable 3D quantitative measures of biological processes in cancellous bone biopsies.     

Imaging lifetime and anisotropy spectra in the frequency domain

We report the development of a system combining the capabilities of fluorescence imaging spectroscopy ( x , λ, I ), fluorescence lifetime (τ) and static and dynamic fluorescence anisotropy ( r ), enabling the wide-field measurement of the spectroscopic parameters of fluorophores: ( x , λ, I , τ, r ). The system employs a frequency domain data collection strategy with a modulated light emitting diode as the light source. A polarization rotator placed in the excitation path after a polarizer allows alternating parallel and perpendicular images to be collected without moving parts. A second polarizer on the emission side serves as the analyzer, leading to estimations of the wavelength-dependent dynamic anisotropies. The spectrograph has a nominal range of 365–920 nm; however, the light-emitting diodes and filter sets used in this study restricted the usable range from about 510 to 700 nm. The system was tested on rhodamine 6G (R6G) solutions containing 0, 15, 37, 45, 59, 74 and 91 glycerol. These experiments gave rotational diffusion results comparing favourably with literature values while also demonstrating a trend towards shorter measured lifetimes at high refractive index. The ability of the system to resolve mixtures was tested on mixtures of anti-human IgG-FITC (γ-chain-specific) and R6G. These fluorophores have similar lifetimes but could be separated using anisotropy parameters. The imaging capabilities of the system were tested on mixtures of fluorescent beads with glycerol solutions of R6G.     

髓腔锉锉齿的设计与研究

设计与研究的主要内容是人工髋关节置换手术中常用的髓腔锉 ,其设计的重点是髓腔锉的齿形及其齿形参数 ,选择合理的齿形及其参数从而达到手术过程中省力和对骨组织损伤小的临床要求。设计的主要成果是找到了一种较为理想的齿形及其对应的齿形参数 ,并通过实验方法得到证实     

Multiscale characterization of pathological bone tissue

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.     

High dose glucocorticoid hampers bone formation and resorption after bone marrow ablation in rat

We analyzed the effect of glucocorticoid on bone regeneration after bone marrow ablation in tibiae of 8-week-old rats. Methylprednisolone sodium succinate (MPSS) was injected intramuscularly at a dose of 100 mg/kg/day for 3 days. Tibiae on days 1, 3, 5, 7, 10, 12, and 14 after ablation were subjected to tartrate-resistant acid phosphatase staining, immunohistochemistry, in situ hybridization, and transmission electron microscopy (TEM), and measurement of the volume of newly-formed bone and the osteoclast number. MPSS significantly decreased the newly-formed bone volume on day 7, and immature bone still remained on day 10 in the MPSS-treated group. The volume of this bone was significantly higher than that in the control group. However, there were no differences between the groups in the osteoclast number, the expression of mRNAs for osteoblast differentiation markers, and alkaline phosphatase and cathepsin K judged by immunohistochemistry. TEM findings showed no difference in the form of osteoblasts, whereas osteoclasts in the MPSS-treated group had less developed ruffled borders, compared to those in the control group. These results suggest that MPSS treatment affects neither the differentiation nor the shape of osteoblasts, and does not change the osteoclast number or the cathepsin K level. However, high dose MPSS inhibits both bone formation and resorption during bone regeneration after rat tibial bone marrow ablation, and inhibits ruffled border formation in osteoclasts. These data will be useful to develop bone regenerative therapies for bone diseases due to high dose steroid administration.     

An investigation of segmentation methods and texture analysis applied to tomographic images of human vertebral cancellous bone

The goal of this study is to determine architectural and textural parameters on computed tomographic (CT) images, allowing us to explain the mechanical compressive properties of bone. Although the resolution (150 μm) is of the same order of magnitude as the trabecular thickness, this method enables the possibility of perfecting an in vivo peripheral CT system with an acceptable radiation dose for the patient. This study was performed on L2 vertebrae cancellous bone specimens taken after necropsy in 22 subjects aged 47–95 years (mean: 79 years). The segmentation process is a crucial point in the determination of accurate architectural parameters. In this paper the use of two different segmentation methods is investigated, based on an edge enhancement and a region growing approach. The images are compared and the architectural parameters extracted from the images segmented by both methods lead to a quantitative evaluation. The parameters are found to be globally robust towards the segmentation process, although some of them are much more sensitive to the approach used. Highly significant correlations ( P  < 0.0005) have been obtained between the two segmentation methods for all the parameters, with ρ ranging from 0.70 to 0.93. In order to improve the assessment of bone architecture, texture analysis (run length method) was investigated. New features are obtained from an image reduced to 16 grey-levels. Textural parameters in addition to architectural parameters in a multivariate regression model increase significantly ( P  = 0.01) the prediction of the maximum compressive strength (variation of r ² from 0.75 up to 0.89).     

Influence of crystal anisotropy on subsurface damage in ultra-precision cylindrical turning of CaF2

An optical microcavity, which stores light at a certain spot, is an essential component to realize all-optical signal processing. Single-crystal calcium fluoride (CaF₂) theoretically shows a high Q-factor which is a desirable optical property. The CaF₂ microcavity can only be manufactured by ultra-precision cylindrical turning (UPCT). The authors have studied UPCT of CaF₂ and shown the influence of crystal anisotropy and tool geometry on surface roughness and subsurface damage. The study indicated that a smaller nose radius of the cutting tool led to shallower subsurface damage. Thus, it is inferred that a smaller nose radius compared to the previous nose radius (0.05 mm) can further reduce subsurface damage. Nevertheless, the mechanism that causes a difference in subsurface damage due to crystal anisotropy is not sufficiently clear. The influence of subsurface damage on microcavity performance is still unclear. In this study, the UPCT of CaF₂ was conducted using a tool with a nose radius of 0.01 mm. The subsurface damage was investigated by transmission electron microscope (TEM) observation from the viewpoint of the change in crystal lattice arrangement. In our previous study, fast Fourier transfer (FFT) analysis was used for confirmation of change of crystal structure. In this study, FFT analysis was also used to quantitatively evaluate the depth of subsurface damage. In addition, inverse fast Fourier transfer (IFFT) was used to analyze change of crystal lattice arrangement clearly, which enables discussion of the influence of slip systems. Finally, optical microcavities are manufactured without any crack, and the influence of subsurface damage on microcavity performance is experimentally evaluated using a wavelength tunable laser and power meter.