多孔β—TCP生物陶瓷骨内植入后的X射线能谱分析

采用两种不同的扫描电镜与Ｘ射线能谱仪测量了多孔磷酸三钙（β－ＴＣＰ）生物陶瓷骨内植入后植入陶瓷,界面和兔股骨的Ｘ射线能谱和元素比,比较了植入后材料以及界面元素比和组成的变化,Ｘ射线能谱结合扫描电镜,拉曼光谱和红外光谱,对植入后磷酸钙生物陶瓷从无生命的材料转为生命骨骼的生物转化作了深入的探讨,为多孔β－ＴＣＰ生物陶瓷的生物降解和骨生成机制提供了更充足的证据。     

Characterization,and antibacterial properties of novel silver releasing nanocomposite scaffolds fabricated by the gas foaming/salt-leaching technique

The main aim of the work was the fabrication of novel silver releasing nanocomposite scaffolds, for bone treatment, by the gas foaming/particulate-leaching technique. Silver doped bioactive glass nanoparticles were used as a filler, to provide the scaffolds with bioactivity, as well as anti-bacterial properties. Nanocomposite scaffolds containing 0, 20 and 40 wt% glass contents were prepared and coded as PAg0, PAg20 and PAg40, respectively. The scaffolds were characterized by SEM/EDXA, FTIR and TGA. Examination of SEM microphotographs showed that, the produced scaffolds had well interconnected structures. For PAg0, PAg20 and PAg40, the maximum pore sizes were about 250, 150 and 100 μm, respectively, while their porosities % were 92%, 89% and 83%, respectively. Degradation studies were carried out, by incubating the scaffolds in simulated body fluid, for a month. Results revealed the possibility to modulate and improve the degradation of the scaffolds by increasing their glass contents. The final weight losses measured for PAg0, PAg20 and PAg40 were 12.76%, 14.61% and 17.42%, respectively. On the other hand, the highest water absorption values recorded for those scaffolds were 61.89%, 240.36% and 270.87%, respectively, indicating that, the addition of glass nanoparticles to the scaffolds improved their water absorption abilities. Both PAg20 and PAg40 induced an apatite layer on their surfaces, had anti-bacterial effect in agar plates, and their silver releasing profiles followed a diffusion-controlled mechanism. Therefore they could be used for bone treatment.     

Interpretation of scanning electron microscopic images of abraded forming bone surfaces

The experimental abrasion of forming bone surfaces was conducted so that such surfaces could be characterized. This is particularly important to bone remodeling studies utilizing scanning electron microscope (SEM) imaging of archeological material. Forming surfaces derived from subadult macaque cranial bone were treated by particle abrasion, water abrasion, sliding abrasion, brushing, manual rubbing, weight, exfoliation, chipping and replication. Acetic acid treatments were also performed. The effects of abrasive agents are specific but generally fall into rough (particle and water abrasion) and smooth (sliding abrasion, brushing, rubbing and weight) categories. Protohistoric human and Plio-Pleistocene hominid subadult craniofacial remains were observed with the SEM for comparison with experimental data. The more recent material appeared smooth, probably as a result of specimen preparation procedures using brushes. Surfaces were still interpretable as forming, however, using a more abrasion-resistant feature called intervascular ridging (IVR) described in this study. The IVR pattern is also recognized on the hominid sample, confirming the possibility of performing remodeling studies on abraded fossil material. The abrasion characteristics are somewhat more difficult to classify, however. Abrasion is defined and discussed relative to remodeling studies and taphonomy. The usefulness of the experimental data reported here, however, in paleoenvironmental reconstruction, has yet to be fully realized. Acid and mechanical preparation techniques are briefly addressed. It is concluded that it is possible to characterize a forming surface as abraded according to the findings of this study and that acid, if handled with care, will more likely preserve microanatomical surface detail. It would also be in everyone's interest to employ a less abrasive cleaning regime on archeological specimens.     

Routine use of backscattered electron imaging to visualize cytochemical and autoradiographic reactions in semi-thin plastic sections.

The scanning electron microscope (SEM) was used to examine cytochemical and autoradiographic reactions in 2-microns semi-thin sections of tissues conventionally fixed and embedded in various resins. The sections were examined using both the secondary and backscatter modes of the SEM at magnifications within the range attainable with the light microscope. Both modes allowed the imaging of phosphatase reaction product using cerium and lead capture, lectin-gold, and immunogold labeling, with and without silver enhancement, and autoradiography. Backscattered electron imaging (BEI), however, provided images with more contrast and structural details. This approach allows examination of large sections, with more contrast and resolution than the light microscope, and visualization of reactions not visible with this instrument. The improved imaging and the simple and conventional preparation of specimens indicate that BEI can be used routinely to examine tissue organization, cell structure, and the content of the various cell compartments with a resolution approaching that of transmission electron microscopy.     

Recent developments in electron microscopical techniques for studying ion localization in plant cells

This paper reviews recent technical approaches to the study of element localization in plant cells. It is concerned with sample preparation; with the use of electron probe microanalysis in the low temperature scanning electron microscope; with the use of electron energy loss spectrocopy and electron spectroscopic imaging in the transmission electron microscope. Basic principles of instrumentation, special problems during cryopreparation of plant tissues, and the application of these techniques within selected fields of botanical interests are briefly discussed.Abbreviations EDXA Energy Dispersive X-Ray Analysis - EELS Electron Energy Loss Spectrometry - EPMA Electron Probe Microanalysis - ESEM Environmental Scanning Electron Microscope - ESI Electron Spectroscopic Imaging - HPF High Pressure Freezing - LTSEM Low Temperature Scanning Electron Microscope - SEM Scanning Electron Micrsocope - TEM Transmission Electron Microscope Botanisches Institut der Tierärztlichen Hochschule HannoverDedicated to Professor André Läuchli on the occasion of his 60th birthday     

Dexamethasone-loaded hydroxyapatite enhances bone regeneration in rat calvarial defects

A combination of bioceramics and osteogenic factors is potentially useful for bone regeneration applications. In the present study, hydroxyapatite particles (HA) were loaded with dexamethasone (Dex) and then characterized using SEM and drug release study. The bone regeneration ability of Dex-loaded HA (Dex/HA) was investigated in a rat critical size bone defect using digital mammography, multislice spiral-computed tomography (MSCT) imaging, and histological analysis. The HA and Dex/HA showed nano and micro-scale morphology with a nearly homogenous distribution of diameter. In addition, about 90 % of the drug was released from Dex/HA over a period of three days. After 8 weeks of implantation in rat calvarial defects, no sign of inflammation or complication was observed at the site of surgery. According to digital mammography and MSCT, Dex/HA showed the highest bone regeneration in rat bone defects compared to those received drug-free HA. Histological studies confirmed these data and showed osteointegration to the surrounding tissue. Taking all together, it was demonstrated that Dex/HA can be used as an appropriate synthetic graft for bone tissue engineering applications. These newly developed bioceramics can be used as new bone graft substitutes in orthopaedic surgery and is capable of enhancing bone regeneration.     

Localization of cadmium in the root cells of <Emphasis Type="Italic">Allium cepa</Emphasis> by energy dispersive x-ray analysis

Allium cepa L. roots were exposed to 0.1 and 1.0 mM Cd for 6, 24 and 48 h and the localization of Cd in the root tissue was investigated. Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDXA) were performed on frozen-dried tissues of roots. No Cd was detected in the roots treated with only 0.1 mM Cd, while after exposure to higher Cd concentration (1.0 mM) Cd was observed in cell wall and in cytoplasm in the epidermis, cortex and vascular tissues in the roots.     

Age changes in bone

Changes in bone structure as a function of age have been studied by simple inspection, x-ray imaging, stereo-photography, deep field optical microscopy, circularly polarised light microscopy, and scanning electron microscopy (SEM), including both topographic and compositional backscattered electron (BSE) imaging modes. The study of bone as a three-dimensional object, rather than in thin sections, enables us to envisage modelling and remodelling processes in context. The study of ultra-flat block surfaces permits the acquisition of data from an effectively very thin layer in the block face, and to examine bone as a spectrum of tissue types varying in the degree of mineralisation. Particular attention has been paid in our earlier studies to the iliac crest, lumbar vertebral bodies, femoral mid-shaft, neck and head and parietal and frontal skull bones. Recently, we have compared findings from these sites with observations on the mandible. We conclude, from our new imaging data, that common generalisations about the changes in bone in ageing and osteoporosis are too simplified, and that the mandible differs sufficiently from post-cranial skeletal sites that it would be unwise to extrapolate from findings in the jaw to the circumstances elsewhere.     

In vivo K-edge imaging with synchrotron radiation.

We present in this paper two imaging techniques using contrast agents assessed with in vivo experiments. Both methods are based on the same physical principle, and were implemented at the European Synchrotron Radiation Facility medical beamline. The first one is intravenous coronary angiography using synchrotron radiation X-rays. This imaging technique has been planned for human studies in the near future. We describe the first experiments that were carried out with pigs at the ESRF. The second imaging mode is computed tomography using synchrotron radiation on rats bearing brain tumors. Owing to synchrotron radiation physical properties, these new imaging methods provide additional information compared to conventional techniques. After infusion of the contrast agent, it is possible to derive from the images the concentration of the contrast agent in the tumor area for the computed tomography and in any visible vessel for the angiography method.     

Osteocyte calcium signaling response to bone matrix deformation

Osteocytes embedded in calcified bone matrix have been widely believed to play important roles in mechanosensing to achieve adaptive bone remodeling in a changing mechanical environment. In vitro studies have clarified several types of mechanical stimuli such as hydrostatic pressure, fluid shear stress, and direct deformation influence osteocyte functions. However, osteocyte response to mechanical stimuli in the bone matrix has not been clearly understood. In this study, we observed the osteocyte calcium signaling response to the quantitatively applied deformation in the bone matrix. A novel experimental system was developed to apply deformation to cultured bone tissue with osteocytes on a microscope stage. As a mechanical stimulus to the osteocytes in bone matrix, in-plane shear deformation was applied using a pair of glass microneedles to bone fragments, obtained from 13-day-old embryonic chick calvariae. Deformation of bone matrix and cells was quantitatively evaluated using an image correlation method by applying for differential interference contrast images of the matrix and fluorescent images of immunolabeled osteocytes, together with imaging of the cellular calcium transient using a ratiometric method. As a result, it was confirmed that the newly developed system enables us to apply deformation to bone matrix and osteocytes successfully under the microscope without significant focal plane shift or deviation from the observation view field. The system could be a basis for further development to investigate the mechanosensing mechanism of osteocytes in bone matrix through examination of various types of rapid biochemical signaling responses and intercellular communication induced by matrix deformation.     

树鼩的骨元和骨细胞穴微观结构超微影像的定性比较

近年来,由于树鼩与灵长类动物的亲缘关系,它们引起了人类发展和疾病研究的兴趣。在这项对树鼩,鼠,狗,狒狒和人类的骨骼超微形态的比较研究中,我们定性分析了骨骼的微观结构和形态,以评估树鼩对人类的亲近程度。在3只成年雄性树鼩 (滇西亚种) (Tupaia belangeri chinensis）的股骨中,使用荧光素异硫氰酸盐样品制备和染色共聚焦成像研究了皮质骨的骨元结构,并使用酸蚀刻SEM观察了骨细胞穴的形态。总体而言,树鼩中骨样形成物的密度和结构以及骨细胞穴的形态更像鼠, 与人类,狗和狒狒都明显不同。这些发现表明,尽管树鼩在系统发育上比鼠更接近人类,但它们的骨骼超微形态仍与鼠接近。这是除狗和狒狒之外,第一次对树鼩的骨元和骨细胞穴进行超微影像的研究。这个比较研究的结果丰富了我们对早期灵长类动物骨骼发育,适应性和进化的理解。未来有必要进行进一步的定量比较研究来表征树鼩骨骼的微观形态。     

Transversely isotropic elasticity imaging of cancellous bone

To measure spatial variations in mechanical properties of biological materials, prior studies have typically performed mechanical tests on excised specimens of tissue. Less invasive measurements, however, are preferable in many applications, such as patient-specific modeling, disease diagnosis, and tracking of age- or damage-related degradation of mechanical properties. Elasticity imaging (elastography) is a nondestructive imaging method in which the distribution of elastic properties throughout a specimen can be reconstructed from measured strain or displacement fields. To date, most work in elasticity imaging has concerned incompressible, isotropic materials. This study presents an extension of elasticity imaging to three-dimensional, compressible, transversely isotropic materials. The formulation and solution of an inverse problem for an anisotropic tissue subjected to a combination of quasi-static loads is described, and an optimization and regularization strategy that indirectly obtains the solution to the inverse problem is presented. Several applications of transversely isotropic elasticity imaging to cancellous bone from the human vertebra are then considered. The feasibility of using isotropic elasticity imaging to obtain meaningful reconstructions of the distribution of material properties for vertebral cancellous bone from experiment is established. However, using simulation, it is shown that an isotropic reconstruction is not appropriate for anisotropic materials. It is further shown that the transversely isotropic method identifies a solution that predicts the measured displacements, reveals regions of low stiffness, and recovers all five elastic parameters with approximately 10% error. The recovery of a given elastic parameter is found to require the presence of its corresponding strain (e.g., a deformation that generates ??? is necessary to reconstruct C????), and the application of regularization is shown to improve accuracy. Finally, the effects of noise on reconstruction quality is demonstrated and a signal-to-noise ratio (SNR) of 40 dB is identified as a reasonable threshold for obtaining accurate reconstructions from experimental data. This study demonstrates that given an appropriate set of displacement fields, level of regularization, and signal strength, the transversely isotropic method can recover the relative magnitudes of all five elastic parameters without an independent measurement of stress. The quality of the reconstructions improves with increasing contrast, magnitude of deformation, and asymmetry in the distributions of material properties, indicating that elasticity imaging of cancellous bone could be a useful tool in laboratory studies to monitor the progression of damage and disease in this tissue.     

Osteoporosis evaluation through full developed speckle imaging

Osteoporosis is a disease characterized by bone mineral density reduction, weakening the bone structure. Its diagnosis is performed using ionizing radiation, increasing health risk. Optical techniques are safer, due to non‐ionizing radiation use, but limited to the analyses of bone tissue. This limitation may be circumvented in the oral cavity. In this work we explored the use of laser speckle imaging (LSI) to differentiate the sound and osteoporotic maxilla and mandible bones in an in vitro model. Osteoporosis lesions were simulated with acid attack. The samples were evaluated by optical profilometry and LSI, using a custom software. Two image parameters were evaluated, speckle contrast ration and patches ratio. With the speckle contrast ratio, it was possible to differentiate sound from osteoporotic tissue. From speckle patches ratio it was observed a negative correlation with the roughness parameter. LSI is a promissory technique for assessment of osteoporosis lesions on alveolar bone.     

Brief communication: Paleohistopathological analysis of pathology museum specimens: Can periosteal reaction microstructure explain lesion etiology?

The assertion that the microstructure of periosteal new bone formation can be used to differentiate between disease etiologies (Schultz: Yrbk Phys Anthropol 44 2001 106–147; Schultz: Identification of pathological conditions in human skeletal remains, 2nd ed. London: Academic Press 2003 73–109) was tested in a pilot‐study, using diagnosed bone specimens from St George's Hospital Pathology Museum, London, UK. Embedded bone specimens exhibiting pathological periosteal new bone formation were examined using scanning electron microscopy in back‐scattered electron imaging mode (SEM‐BSE). The results suggest that several histological features (i.e. Grenzstreifen, Polsters, and sinuous lacunae) deemed to be diagnostic of specific pathological conditions are of no specific diagnostic value, as they are encountered in pathological conditions of differing disease etiology. These results tie in with a previous investigation demonstrating a lack of diagnostic qualitative or quantitative characteristics seen in the macroscopic and radiographic appearance of periosteal reactions (Weston: Am J Phys Anthropol 137 2008 48–59). Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

The 18 kDa translocator protein (peripheral benzodiazepine receptor) expression in the bone of normal, osteoprotegerin or low calcium diet treated mice

The presence of the translocator protein (TSPO), previously named as the mitochondrial or peripheral benzodiazepine receptor, in bone cells was studied in vitro and in situ using RT-qPCR, and receptor autoradiography using the selective TSPO ligand PK11195.In vitro, the TSPO is highly expressed in osteoblastic and osteoclastic cells.In situ, constitutive expression of TSPO is found in bone marrow and trabecular bone, e.g., spongiosa. Mice with a reduction of bone turnover induced by a 4-day treatment of osteoprotegerin reduces [(3)H]PK11195 binding in the spongiosa (320±128 Bq x mg(-1), 499±106 Bq x mg(-1) in saline-treated controls). In contrast, mice with an increase in bone turnover caused by a 4-day low calcium diet increases [(3)H]PK11195 binding in the spongiosa (615±90 Bq x mg(-1)). Further, our study includes technical feasibility data on [(18)F]fluoride microPET imaging of rodent bone with altered turnover. Despite [(18)F]fluoride having high uptake, the in vivo signal differences were small. Using a phantom model, we describe the spillover effect and partial volume loss that affect the quantitative microPET imaging of the small bone structures in experimental mouse models. In summary, we demonstrate the expression of TSPO in small rodent bone tissues, including osteoblasts and osteoclasts. A trend increase in TSPO expression was observed in the spongiosa from low to high bone turnover conditions. However, despite the potential utility of TSPO expression as an in vivo biomarker of bone turnover in experimental rodent models, our small animal PET imaging data using [(18)F]fluoride show that even under the condition of a good biological signal-to-noise ratio and high tracer uptake, the currently achievable instrument sensitivity and spatial resolution is unlikely to be sufficient to detect subtle differences in small structures, such as mouse bone.     

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.     

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging,Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.For this purpose, we injected 10⁵ MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg¹. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula¹. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified^2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions^5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles⁷.In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.     

Development of contrast agents targeted to macrophage scavenger receptors for MRI of vascular inflammation

Atherosclerosis is a leading cause of death in the U.S. Because there is a potential to prevent coronary and arterial disease through early diagnosis, there is a need for methods to image arteries in the subclinical stage as well as clinical stage using various noninvasive techniques, including magnetic resonance imaging (MRI). We describe a development of a novel MRI contrast agent targeted to plaques that will allow imaging of lesion formation. The contrast agent is directed to macrophages, one of the earliest components of developing plaques. Macrophages are labeled through the macrophage scavenger receptor A, a macrophage specific cell surface protein, using an MRI contrast agent derived from scavenger receptor ligands. We have synthesized and characterized these contrast agents with a range of relaxivities. In vitro studies show that the targeted contrast agent accumulates in macrophages, and solution studies indicate that micromolar concentrations are sufficient to produce contrast in an MR image. Cell toxicity and initial biodistribution studies indicate low toxicity, no detectable retention in normal blood vessels, and rapid clearance from blood. The promising performance of this contrast agent targeted toward vascular inflammation opens doors to tracking of other inflammatory diseases such as tumor immunotherapy and transplant acceptance using MRI.     

Multimodality Imaging of Tumor and Bone Response in a Mouse Model of Bony Metastasis

Cancer drug development generally performs in vivo evaluation of treatment effects that have traditionally relied on detection of morphologic changes. The emergence of new targeted therapies, which may not result in gross morphologic changes, has spurred investigation into more specific imaging methods to quantify response, such as targeted fluorescent probes and bioluminescent cells. The present study investigated tissue response to docetaxel or zoledronic acid (ZA) in a mouse model of bony metastasis. Intratibial implantations of breast cancer cells (MDA-MB-231) were monitored throughout this study using several modalities: molecular resonance imaging (MRI) tumor volume and apparent diffusion coefficient (ADC), micro-computed tomography (µCT) bone volume, bioluminescence imaging (BLI) reporting cancer cell apoptosis, and fluorescence using Osteosense 800 and CatK 680-FAST. Docetaxel treatment resulted in tumor cell kill reflected by ADC and BLI increases and tumor volume reduction, with delayed bone recovery seen in µCT prefaced by increased osteoblastic activity (Osteosense 800). In contrast, the ZA treatment group produced similar values in MRI, BLI, and Osteosense 800 fluorescence imaging readouts when compared to controls. However, µCT bone volume increased significantly by the first week post-treatment and the CatK 680-FAST signal was slightly diminished by 4 weeks following ZA treatment. Multimodality imaging provides a more comprehensive tool for new drug evaluation and efficacy screening through identification of morphology as well as function and apoptotic signaling.     

Assessment of the bilateral asymmetry of human femurs based on physical,densitometric, and structural rigidity characteristics

The purpose of this study was to perform a comprehensive geometric, densitometric, biomechanical, and statistical analysis of paired femurs for an adult population over a wide age range using three imaging modalities to quantify the departure from symmetry in size, bone mineral density, and cross-sectional structural rigidities.Femur measurements were obtained from 20 pairs of cadaveric femurs. Dimensions of these anatomic sites were measured using calipers directly on the bone and plain radiographs. Dual energy X-ray absorptiometry was used to measure bone mineral density. Bone mineral content and axial and bending rigidities were determined from the CT imaging.No differences were observed between the geometric measurements, DXA based bone mineral density and axial and bending rigidities of left and right femurs (P>0.05 for all cases). Left and right proximal femurs are not significantly different based on geometric, densitometric, and structural rigidity measurements. However, absolute left–right differences for individual patients can be substantial. When using the contralateral femur as a control, the number of femur pairs required to assess significant changes in anatomic dimensions and structural properties induced by a tumor, infection, fracture, or implanted device can range from 3 to 165 pairs depending on the desired effect size or sensitivity (5% or 10% difference).This information is important both for femoral arthroplasty implant design and the use of the contralateral femur as an intra-subject control for clinical assessment and research studies. In addition, our statistical analysis provides sample size estimates for planning future orthopedic research studies.