Daniela Quacci

Electron microscopic visualization of proteoglycans with Alcian Blue

SynopsisThe intercellular matrices of bovine nasal cartilage, chick embryo perichordal cartilage and chick embryo mesenchymal cells culturedin vitro have been examined by electron microscopy after staining them with Alcian Blue in salt solutions according to Scott & Dorling (1965).Matrix granules, which are typical components of cartilage at the ultrastructural level, are not visible after Alcian Blue staining and are replaced by alcianophilic rod-like particles, varying in length and width. With tissue cultures, Alcian Blue stains 40–120 Å chick filaments which display an orthogonal and longitudinal relationship to collagen fibrils.We assume that cartilage matrix granules represent linear proteoglycans that are coiled as a consequence of the usual glutaraldehyde-osmium fixation. It is thought that Alcian Blue, on the other hand, contributes to the stabilization of the proteoglycans in their original structural arrangement. This stabilizing property presumably also results in the sharp visualization of fine filaments in the tissue culture matrix.

Anatomy of the intracortical canal system: scanning electron microscopy study in rabbit femur.

The current model of compact bone is that of a system of longitudinal (Haversian) canals connected by transverse (Volkmann’s) canals. Models based on histology or microcomputed tomography lack the morphologic detail and sense of temporal development provided by direct observation. Using direct scanning electron microscopy observation, we studied the bone surface and structure of the intracortical canal system in paired fractured surfaces in rabbit femurs, examining density of canal openings on periosteal and endosteal surfaces, internal network nodes and canal sizes, and collagen lining of the inner canal system. The blood supply of the diaphyseal compact bone entered the cortex through the canal openings on the endosteal and periosteal surfaces, with different morphologic features in the midshaft and distal shaft; their density was higher on endosteal than on periosteal surfaces in the midshaft but with no major differences among subregions. The circumference measurements along Haversian canals documented a steady reduction behind the head of the cutting cone but rather random variations as the distance from the head increased. These observations suggested discontinuous development and variable lamellar apposition rate of osteons in different segments of their trajectory. The frequent branching and types of network nodes suggested substantial osteonal plasticity and supported the model of a network organization. The collagen fibers of the canal wall were organized in intertwined, longitudinally oriented bundles with 0.1- to 0.5-μm holes connecting the canal lumen with the osteocyte canalicular system.

Morphometry and Patterns of Lamellar Bone in Human Haversian Systems

The lamellar architecture of secondary osteons (Haversian systems) has been studied with scanning electron microscopy (SEM) in transverse sections of human cortical bone. Na3PO4 etching was used to improve the resolution of the interface between neighboring lamellae and the precision of measurements. These technical improvements permitted testing of earlier morphometry assumptions concerning lamellar thickness while revealing the existence of different lamellar patterns. The mean lamellar thickness was 9.0 ± 2.13 μm, thicker and with a wider range of variation with respect to earlier measurements. The number of lamellae showed a direct correlation with the lamellar bone area, and their thickness had a random distribution for osteonal size classes. The circular, concentrical pattern was the more frequently observed, but spiral and crescent‐moon‐shaped lamellae were also documented. Selected osteons were examined by either SEM or SEM combined with polarized light microscopy allowing comparisons of corresponding sectors of the osteon. The bright bands observed with polarized light corresponded to the grooves observed in etched sections by SEM. The dark bands corresponded to the lamellar surface with the cut fibrils oriented approximately longitudinally along the central canal axis. However, lamellae with large and blurred bright bands could be observed, which did not correspond to a groove observed by SEM. These findings are in contrast with the assumption that all the fibril layers within a lamella are oriented along a constant and unchangeable angle. The different lamellar patterns may be explained by the synchronous or staggered recruitment and activation of osteoblasts committed to the osteons completion. Anat Rec, 2012.

Atherosclerotic alterations in human carotid observed by scanning electron microscopy

Atherosclerosis involves all the layers of the artery wall, but the events involving the intimal portion are fundamental to understand the evolution and gravity of lesions. This study shows that scanning microscopy is instrumental for better understanding the physiopathology of this disease.

Ultrastructure of periprosthetic Dacron knee ligament tissue: Two cases of ruptured anterior cruciate ligament reconstruction

Light- and electron-microscopic investigations were performed on two failed Dacron ligaments that had been removed from 2 patients shortly after failure of the implant 2-3 years after reconstruction of the anterior cruciate ligament. Two different cell populations and matrices were correlated with closeness to the Dacron threads. Fibroblasts surrounded by connective tissue with collagen fibrils were located far from the Dacron threads. Roundish cells, appearing to be myofibroblasts surrounded by a more lax connective tissue and elastic fibers, were found close to the Dacron threads. The presence of myofibroblasts and the matrix differentiation could be attributed to the different mechanical forces acting on the Dacron and on the connective tissue because of their different coefficients of elasticity. The sparse occurrence of inflammatory cells in the synovial membrane and in the connective tissue surrounding the Dacron supports the biologic inertness of this artificial material. However, the repair tissue was not structured to resist tension stresses.

Laminar Pattern of Mineral Calcium-Phosphorus Deposits in a Human Carotid Plaque Nanoscale Ultrastructure and Elemental Analysis

Atherosclerotic calcifications are related to poor prognosis and all-cause mortality in large population studies.1 Moreover, vascular calcifications are inversely associated with the potential reduction of plaque volume in regression studies during statin treatment, which suggests that more calcified lesions are less likely to undergo positive remodeling.2 Recently, it has been suggested that calcification is a tightly regulated process of mineralization akin to bone formation.3 In plaques, the deposits consist of a nonhomogeneous composite that contains hydroxyapatite mineral nanocrystals embedded in a collagenous organic matrix, whereas at a nanoscale level, apatite crystals interact with cholesterol crystallites. In this context, mineralization may result from a basic template pattern generated by the organic …

Chemical etching in processing cortical bone specimens for scanning electron microscopy.

Transverse and longitudinal sectioning of undecalcified cortical bone is a commonly employed technique for investigating the lamellar structure of the osteons. Since a flat surface is required, the specimen has to be grinded and then polished. Whereas the smear of debris and inorganic/organic deposits left by these treatments cannot be removed by ultrasonication alone, a chemical treatment of the specimen surface with either a basic or an acid etching solution is currently employed. A further effect of the latter can be the enhancement of the lamellar bone pattern. The kind of etching solution, its pH, the concentration of etchants, and the contact time significantly affect the sectioned surface when it is observed with scanning electron microscopy (SEM). The etching procedures can severely influence the obtained images. Homogeneous cortical bone specimens were sampled from the first metatarsal of two fresh human subjects. One or two cut surfaces were exposed to different acid and basic solutions in bonded conditions. Considering the type of chemical agents, the solution pH, and the exposure time of the specimens, the effects of several etching media have been investigated and compared. Strong etching, either acid or basic produced surface decalcification and severe damage of the collagen matrix, compromising any morphological or morphometric analysis. Weak acid etching (for example citric and acetic acid), even though causing distinctive alteration of the sample, enhanced the visibility of the lamellar pattern, while the polyphosphate treatment of the surface decalcified a thin layer matrix, ensuring a good visibility of fibrils and avoiding rough distortions. Microsc. Res. Tech. 77:653–660, 2014.

Scanning Electron Microscopy in forensic investigations: More views from more applications

The purpose of this presentation is to expand and highlight the range of applications of scanning electron microscopy to forensic science, following the overview which was shown last year at the LXX SIAI Congress. All examples shown of forensic uses of SEM were carried out over the last few years at the Human Morphology Laboratory of the University of Insubria. These studies include: - The identification and characterization of different toolmarks found on human bones. Some toolmarks have a distinctive morphology and allow a reliable identification of the weapon or instrument used. For this purpose, we will illustrate a few examples of dismemberment with different types of saw and will show the peculiar bone patterns left by different cutting edges (knives, axes, cutters ...); - The examination of human tissues and of medical devices (catheters etc.) for the early detection and identification of slow-growing microorganisms (e.g. some fungi). The diagnosis of these microorganisms would have otherwise required molecular biology techniques, which are not only expensive but also not always available or applicable in the field of forensics (for instance, when the specimen is inadequate for external contaminations or is into a state of conservation far from optimal), or conventional cultures in vitro, which require much longer times and may be easily spoiled by inopportune drug administration; - The use of scanning electron microscopy and of X-ray spectroscopy as auxiliary and “creative” tools to discover mystifications and frauds against insurance companies.