Marcella Reguzzoni

Endothelin and nitric oxide synthase in lymphatic endothelial cells: immunolocalization in vivo and in vitro.

Endothelin (ET) is an endothelium‐derived multifunctional peptide that produces a potent, long‐lasting vasoconstriction. Nitric oxide (NO), besides being the most important endothelium‐derived relaxant factor in blood vessels, is supposed to be involved in regulating the interactions among endothelium, adhesive molecules, and leukocytes.

Immunohistochemical localization of lipoperoxidation products in normal human placenta

4-Hydroxynonenal (4-HNE) is a major propagation product of lipid peroxidation that is supposed to be responsible for some of the effects associated with oxidative stress in tissues. We have investigated the possible occurrence and distribution of 4-HNE-immunoreactivity in human normal placenta using immunocytochemistry. Specific immunostaining was observed in cytotrophoblast cells, syncytiotrophoblast, some cells of the villous mesenchyme and some endothelial cells of first trimester and term placentae. The detection of 4-HNE-immunoreactivity in placenta raises the question whether lipoperoxidation products are produced locally in placental cells or represent exogenous products that derive from maternal blood flow. Since trophoblastic cells and villous macrophages are provided by a scavenger receptor, it is conceivable that these cells may play a protective role with regard to the diffusion of lipoperoxidation products from the mother to the embryo. However, since a significant degree of lipid oxidative modification does not take place in plasma, it is presumed that 4-HNE is a local product of placental metabolism. In line with this hypothesis, it is proposed that maternal low density lipoproteins, which are the major source of cholesterol for placental steroid synthesis, might be oxidized by villous cells during their traversal through the villous wall.

Not only tendons: The other architecture of collagen fibrils

For many decades the fibrillar collagens have been the subject of a remarkable body of ultrastructural research. The vast majority of the studies, however, were carried out on tendon or on tendon-derived material. For many reasons this reflects an obvious choice but at the same time it also is an unfortunate circumstance, because this flooding of tendon-related data can easily encourage the false confidence that all connective tissues are similar. The reality is quite the opposite, and a different fibrillar structure has been long time observed on collagen fibrils from different tissues, the most notable example being offered by corneal fibrils. The same architecture can be found in a number of disparate tissues and may actually be the prevalent one on a whole-body scale. Although these fibrils diverge from those of tendon in their architecture, size, D-period, composition, cross-linking and fibrillogenesis mechanism, their structure was the subject of rather sparse ultrastructural studies and even today their mere existence is often overlooked or ignored. This paper summarizes the main aspects of the structural biology of these forgotten fibrils.

Presence of calcified tissue in the human temporomandibular joint disc.

Large calcified areas were observed in the articular discs of the temporomandibular joint from five patients suffering from articular dysfunctions. The calcified regions were always located inside the fibrous tissue of the discs. They had a woven bone-like morphological pattern and consisted of a compact mineralized tissue containing cells in irregular lacunae. In all the samples the calcified tissue was completely surrounded by a mineralizing border rich in cells and variously arranged collagen fibrils. Energy-dispersive spectrometry showed that mineralized regions contained large amounts of Ca and P. X-ray powder diffraction identified the crystals in these areas as hydroxyapatite.

Mineralization-related modifications in the calcifying tendons of turkey (Meleagris gallopavo)

The tendons of some birds undergo a physiological process of gradual mineralization, usually limited to the central portion of the tendon and resulting in an increase of the elastic modulus and the ultimate strength. The present study was carried out by light microscopy and scanning electron microscopy and was focused on the structural and ultrastructural modifications occurring in this tissue during biomineralization. In comparison with most other tendons, turkey tendons appeared to be more finely subdivided into thinner fascicles and to contain a greater amount of cell-rich endotenon tissue. The most obvious finding, however, was the complete disappearance of the crimps in the calcified portions of the tendon, while they were present with the usual morphology in the non-mineralized portion. The electron microscopy revealed in the mineralized tendon traces of pre-existing crimps, locked in the straightened-out position by the infiltrating mineral phase. This latter was composed of two different types of fine particles, respectively, growing inside and around the collagen fibrils and appearing as tightly packed platelets or as larger, flat platelets regularly arranged in phase with the D-period of collagen. The perifibrillar mineral could play a critical role in the mechanical coupling of adjoining fascicles and in the transmission of tensile loads along the tendon itself.

3D culture of multipotent cells derived from waste human ovarian follicular liquid and seeded onto gelatin cryogel

Current tissue engineering uses 3D biomaterials in combination with stem cells, since mature cells are often not available in sufficient amounts or quality. Biomaterial scaffolds have been widely used in reconstructive bone surgery not only as cell carriers providing mechanical support, but also as promoters of cell attachment and proliferation (1). In particular, gelatine cryogel scaffolds are promising new biomaterials owing to their biocompatibility and to substain the differentiation of mesenchymal stromal stem cells (MSCs) (2). Human MSC proliferate onto the surfaces with fibroblastic morphology and can differentiate into osteoblasts, chondrocytes and adipocytes (3). These cells can be isolated from several sources, including bone marrow and adipose tissue (4). Our previously studies showed the possibility to obtain MSCs also from the human ovarian follicular liquid (FL) that is usually wasted during in vitro fertilization (5). In this study, we tested the ability of these FL cells to grow and differentiate on gelatine cryogel in comparison with MSCs derived from human bone marrow. Samples and controls were analyzed with confocal and scanning electron microscopes. Results demonstrated that FL cells could grow on the biomaterial not only on the top but also in the layers below till 60mm of deepness. Data suggested that the observed cells are mesenchymal since positive for vimentin and CD44 (a typical MSC marker). Preliminary results showed also the capability of induced FL cells to osteogenic differentiation to produce bone extracellular matrix, expressing some specific proteins (i.e.osteopontin). In conclusion, MSCs derived from waste human ovarian follicular liquid showed promising affinity with 3D gelatine cryogel, opening new potential developments in biotech and medical applications.

The byssus threads of Pinna nobilis: a histochemical and ultrastructural study

The byssus of Pinna nobilis, the largest bivalve mollusc in the Mediterranean Sea, was investigated by histochemistry, immunohistochemistry, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). At low magnification, the byssus threads appeared distinctively elliptical in cross-section, with a typical size approaching 50 x 25 micron and a featureless glassy appearance. Histochemical and immunohistochemical techniques confirmed the presence of elastic domains but the absence of collagen, which is known to be the main component in other molluscs. Ultrastructural analysis by TEM revealed the presence of at least two components within the thread, and an inner arrangement of straight, tightly packed longitudinal streaks. SEM observations while confirming the inner packing of straight, parallel subfibrils, suggested in the fracture surfaces the presence of unidentified substance which cemented together the same subfibrils and which was removed by exposure to extreme pH values. AFM micrographs added further evidence for the tight packing of subfibrils and provided some evidence of orthogonal, barely visible connecting structures. Finally, HCl or NaOH treatment left the subfibrils clean and free from any other component.Â.

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.

The quest for the third dimension: from the Electron Microscope to the 3D printer

Conventional light microscopy (LM) and transmission electron microscopy (TEM) are meant to image planar sections, i.e. bidimensional specimens, and are therefore constrained into a bidimensional world. In contrast, the scanning probe microscopy (SPM) and scanning electron microscopy (SEM) are able to image surfaces, i.e. three-dimensional subjects. Of these techniques, SPM has the additional advantage of directly obtaining three-dimensional datasets from three-dimensional specimens, although this ability is seldom exploited. The SEM is per se limited to 2D pictures of 3D subjects, but its flexibility and performance make possible to re-obtain the third dimension indirectly. A first, simple, time-proven approach is stereophotography. This makes possible an immediate visual appreciation of depth and volume but does not allow quantitative measurements. A subsequent approach is represented by shape-from-stereo reconstruction, which builds a quantitative computer model of the specimen. This is now a consolidated technique and several solutions, both hardware- and software-based, are readily available. Although limited to the development of 2 ½ dimensions, rather than real 3D, this technique is simple and effective and for several years the authors have used a proprietary package [1] featured in a number of published papers. More recently a new generation of shape-from-motion or shape-from-video photogrammetric software [2] makes possible the full recovery of the third dimension, complete with undercuts and texture mapping. All these techniques are now complemented and extended by the availability of inexpensive three-dimensional printers. Going beyond visual appreciation and beyond computer graphics, this technique makes possible to obtain a tangible, material model of the specimen. 3D printing is already in use for educational purposes but can be effectively deployed also in morphological research, making possible to obtain highly magnified, accurate copies of microscopic structures such as molecules, cells and interfaces, adding to the visual appreciation the immediacy of the tactile experience. A few examples are shown.

Different eyes, different views. Scanning Electron Microscopy applied to forensic investigations

The Scanning Electron Microscope (SEM) is an extremely versatile instrument, essential in a wide array of applications in forensic analysis: for example, it is used to analyze gunshot residue, bullet fingerprints, bullet wipe or patterns around the bullet hole, to examine traces of foreign material embedded in or adhered to bullets (which can provides critical information in the trajectory reconstruction of spent bullets); to study environmental dusts, fibers (both natural and artificial) and to identify unknown small particles; to detect non visible blood stains; to analyze diatoms in drowning cases; and for ink and paper analysis. One central feature of SEM is its ability of providing both panoramic and highly magnified views of the same sample, giving an almost 3D view of the specimen. It is the ideal trait d’union between macroscopic information collected during autoptic or investigative activity and microscopic information obtained with the light microscope. Above all, SEM allows performing a progressive and targeted microdissection of the sample. In this presentation, a selected number of investigations are shown in order to illustrate through specific cases general purpose applications. An elderly man was killed with several blows of axe at the head. SEM investigation allowed us to reconstruct the sequence of the blows, to recognize the type of weapon, to determine how this latter was used and how sharp it was. These results allowed the police to reject the initial version of the suspected, which was eventually convicted of willful murder. A young man died with multiple traumatic and fulguration lesions. SEM analysis allowed us to perform a detailed study of the burnt tissue and to reconstruct the path of the electric discharge, concluding that the primary causa mortis was an accidental electrocution, which caused the subsequent trauma. A child died of a sudden, dramatic internal bleeding. The autopsy revealed that some time before she had swallowed a coin battery which had become lodged in the oesophagus. Here the decaying products of the battery caused an electro-chemical dissection of the oesophagus and, finally, of the descending aorta. The SEM analysis revealed the details of the progressive degeneration of the surrounding tissues.