Analysis of living cells grown on different titanium surfaces by time-lapse confocal microscopy.

In this study we have combined fluorescence- and reflection-confocal laser scanning microscopy for the simultaneous visualization of living cells and surface topography beneath them. To this purpose we have designed a specific flow chamber and we have tested it with osteoblasts grown on an opaque, thick support, made of smooth or sandblasted titanium. Cells were loaded with Calcein-AM or tetramethylrhodamine methyl ester (TMRM), two probes employed as indicators of cell viability/morphology and mitochondrial membrane potential, respectively. Besides the acquisition of stacks of confocal sections, the system allowed also vertical views and faithful three-dimensional reconstruction of the samples. Confocal microscope implemented with our flow chamber proved to be a promising tool for time-lapse investigation of cell-biomaterial interactions.

3D analysis of SEM images of corrosion casting using adaptive stereo matching.

The corrosion casting method represents one of the most widely used technique to study the 3D microvascularization of many tissues, both in their normal and pathological conditions. For a long time this technique was used only to perform a qualitative evaluation of the images obtained by scanning electron microscopy (SEM). A quantitative evaluation of vascular parameters (e.g., interbranching and intervascular distances, angle measurements, lengths and diameters) was lacking, mainly because of the difficulties found in the measurement performed on 2D SEM images. Then, some authors reported a quantitative method based on the analyses of stereo-pair images that allowed precise morphometric measurements. To visualize the specimens in 3D, it was necessary to use red-green glasses. In this article we describe a new approach by which we can automatically obtain a 3D reconstruction of vascular cast specimen's surface directly from stereo-images. Moreover, we developed a software that performed micrometric measurements on the 3D construct generated from the stereo-pictures. In conclusion, implementing together these two softwares and applying them to corrosion casting samples made it possible to render in 3D the surface of corrosion cast as well as make quantitative measurements on the corrosion casts.

In situ hybridization by scanning electron microscopy for painting, centromeric, and YAC localization.

The hybridization site of a DNA probe was detected using a scanning electron microscope (SEM), modifying the standard in situ hybridization (ISH) method. The experiments were performed on human metaphases obtained from lymphocyte cultures of human peripheral blood. The libraries and probes used were: 1-chromosome library for the painting of chromosome 1 (wcp 1), an alphoid centromere-specific probe of chromosome 8 (pZ8.4), and the yeast artificial chromosome (YAC) 964-C10 mapped at band p13 on chromosome 12. These probes were labeled by nick translation with biotin and displayed with a gold-conjugated anti biotin goat antibody. The gold signal was amplified by silver enhancement. The chromatides appeared as packages of thin filaments 120 nm high; some of them collapsed, probably due to ISH procedures. All the probes were clearly detected as small gold particles grouped on the surface of the target chromosomes and chromosome sites. Thus, this procedure is useful to clarify the positional relationship between the chromatin filaments and the probe.

Plexiform vascular structures in the human digital dermal layer: a SEM--corrosion casting morphological study.

This study aimed to describe the impressive diversity of vascular plexiform structures of the hypodermal layer of human skin. We chose the human body site with the highest concentration of dermal corpuscles, the human digit, and processed it with the corrosion casting technique and scanning electron microscopy analysis (SEM). This approach proved to be the best tool to study these microvascular architectures, free from any interference by surrounding tissues. We took high-definition pictures of the vascular network of sweat glands, thermoreceptorial and tactile corpuscles, the vessels constituting the glomic bodies and those feeding the hair follicles. We observed that the three-dimensional disposition of these vessels strictly depends on the shape of the corpuscles supplied. We could see the tubular vascularization of the excretory duct of sweat glands and the ovoid one feeding their bodies, sometimes made up of two lobes. In some cases, knowledge of these morphological data regarding the normal disposition in space and intrinsic vascularization structure of the dermal corpuscles can help to explain many of the physiopathological changes occurring during chronic microangiopathic diseases.

The 3D structure of the human urinary bladder mucosa: a scanning electron microscopy study.

We performed a scanning electron microscopy study on the human urinary bladder tunica mucosa. Specimens from bladder biopsies were treated with OsO4 maceration and 1N NaOH maceration methods prior to SEM observation to disclose the three-dimensional organization of the lamina propria, basal lamina and urothelium. The lamina propria housed a well developed capillary plexus just below the basal lamina; the urothelium presented a typical three-layered organization with basal, intermediate and superficial cells. The intermediate cells appeared essentially similar to basal cells in their external features and stretched from the basal lamina up to the superficial layer. The most superficial cells appeared consistently flattened and interconnected by extensive junctional complexes. They showed a peculiar specialization, their apical plasmalemma being thickened with distinctive, stiff plaques, in contrast with the underlying globular or spindle-shaped cells whose plasmalemma was only covered by short microvillosities.

Microvascularization of the human digit as studied by corrosion casting.

The aim of this study was to describe microcirculation in the human digit, focusing on the vascular patterns of its cutaneous and subcutaneous areas. We injected a functional supranumerary human thumb (Wassel type IV) with a low-viscosity acrylic resin through its digital artery. The tissues around the vessels were then digested in hot alkali and the resulting casts treated for scanning electron microscopy. We concentrated on six different areas: the palmar and dorsal side of the skin, the eponychium, the perionychium, the nail bed and the nail root. On the palmar side, many vascular villi were evident: these capillaries followed the arrangement of the fingerprint lines, whereas on the dorsal side they were scattered irregularly inside the dermal papillae. In the hypodermal layer of the palmar area, vascular supports of sweat glands and many arteriovenous anastomoses were visible, along with glomerular-shaped vessels involved in thermic regulation and tactile function. In the eponychium and perionychium, the vascular villi followed the direction of nail growth. In the face of the eponychium in contact with the nail, a wide-mesh net of capillaries was evident. In the nail bed, the vessels were arranged in many longitudinal trabeculae parallel to the major axis of the digit. In the root of the nail, we found many columnar vessels characterized by multiple angiogenic buttons on their surface.

Detachment of titanium and fluorohydroxyapatite particles in unloaded endosseous implants.

The shape, surface composition and morphology of orthopaedic and endosseous dental titanium implants are key factors to achieve post-surgical and long-term mechanical stability and enhance implant osteointegration. In this study a comparison was made between 12 titanium screws, plasma-spray-coated with titanium powders (TPS), and 12 screws with an additional coating of fluorohydroxyapatite (FHA-Ti). Screws were implanted in the femoral and tibial diaphyses of two mongrel sheep and removed with peri-implant tissues 12 weeks after surgery. The vibrational spectroscopic, ultrastructural and morphological analyses showed good osteointegration for both types of implants in host cortical bone. The portion of the FHA-Ti implants in contact with the medullary canal showed a wider area of newly formed peri-implant bone than that of the TPS implants. Morphological and EDAX analyses demonstrated the presence of small titanium debris in the bone medullary spaces near the TPS surface, presumably due to the friction between the host bone and the implant during insertion. Few traces of titanium were detected around FHA-Ti implants, even if smaller FHA debris were present. The present findings suggest that the FHA coating may act as a barrier against the detachment of titanium debris stored in the medullary spaces near the implant surface.

The microvasculature of the lateral choroid plexus in the rat: a scanning electron microscopy study of vascular corrosion casts.

The three-dimensional microvascular structure of many organs can be adequately investigated only using the corrosion casting technique. We applied this method, consisting of an injection of low viscosity acrylic resin through the major vessels and the subsequent digestion of the organic component with strong alkali or acids, to the choroid plexus of the lateral ventricles in the rat, focusing on its structural vascular features. This approach allowed a qualitative morphological description of the choroid plexus of the lateral ventricles, revealing several aspects of their capillary architecture as well as the morphological details underlying its main functional activity, essential to cerebrospinal fluid turnover. Observation of the casts with scanning electron microscopy gave a detailed picture of the choroid plexus of the lateral ventricles of the rat and enabled us to distinguish four different regions, depending on the site that the lateral ventricles occupied: the anterior olfactory region, the main central region, the longer branch and the inferior horn. Each region mostly consisted of spiral capillaries and had specific characteristics. At high magnification, the casts revealed distinctive vascular specializations, such as numerous bulges regularly placed on the capillaries. This morphological investigation underpins a better comprehension of the pathological mechanisms involving the choroid plexus in the lateral ventricles.

Hierarchical structures in fibrillar collagens.

The collagen family includes several large transcripts, usually exceeding 1000 amino acid residues per single chain. As a group, they make up 1/3 of all the protein of the body and are responsible for modelling the framework of connective tissues; individually, they show both a wide variety and a complex hierarchy of mutual interactions, and form a range of functional aggregates including a variety of fibrils, microfibrils and basal membranes. Of the collagens, the fibril-forming types (i.e. the types I, II III, V and XI) are the most abundant and the most extensively studied. At the primary structure level, the amino acid sequence of all collagens is now known in detail and it shows a distinctive domain organization, its composition being dominated by the amino acid glycine (roughly 1/3 of all residues) and by post-translational hydroxylation of proline and lysine residues. Collagen secondary and tertiary structure, which together give origin to a classic triple helix, were painstakingly determined in the 1950s and 1960s. In contrast with the primary, secondary and tertiary structure, the supramolecular arrangement within collagen fibres seems to be far more elusive, and none of the models so far advanced can be said to be universally accepted. Half a century of research and debate spawned numerous mutually incompatible models, most of them focussing either on a quasi-crystalline supramolecular array or on several forms of microfibrillar aggregates, while radial fibrils, epitaxial fibrils and other structural models have almost been ignored. In many cases, data gained with a single technique from a single tissue were arbitrarily given a general legitimacy, whilst other well-documented morphological evidence went virtually unnoticed by the scientific community.Moreover, in recent years there has been a growing interest in the multiple interactions of collagens with the other macromolecules of the extra-cellular matrix, as their structure and their functional role become known. It is now indisputable that collagen interacts and forms functional entities with several other macromolecules of the extracellual matrix. This paper will succinctly review some current concepts on the structural biology of collagen higher-order structures.

Electromagnetic stimulation on the bone growth using backscattered electron imaging.

The events at the hydroxyapatite implant material/tissue interface following electromagnetic stimulation were studied in the rabbit. Two kinds of hydroxyapatite were used: natural (NA) and synthetic (HA) both with a grain size of <50 microm. Bone defects, artificially created in rabbit tibiae, were filled with the material examined. One group of animals was exposed immediately after surgery and every 12h thereafter to 30-min treatments with electromagnetic fields (PEMFs). A second group was used as a control (untreated). Two and 4 weeks after implantation, animals were sacrificed and bone samples processed for LM, TEM and SEM using a backscatter electron detector for the evaluation of bone growth. This study indicates that HA has more osteoconductivity than NA, and shows that PEMF-treatment results in a benefit in accelerating bone formation at early time periods.

Tapping-mode atomic force microscopy in fluid of hydrated extracellular matrix.

Fragments of native, hydrated rat tail tendon were imaged by tapping-mode atomic force microscopy while immersed in fluid. The specimens were soft and sensitive to the operating parameters, and with minimal imaging pressure the collagen fibrils appeared covered by irregular blobs or by filamentous material. A slight increase in pressure caused the underlying fibril surface to appear, with an evident D-period, gap- and overlap-zones and three intraperiod ridges. Fibrils often ran parallel and in phase, implying some coupling mechanism. Longitudinal subfibrils, 8-9 nm thick, occasionally appeared. The simultaneous acquisition of the "tapping amplitude" along with the usual "height" channel clearly confirmed the presence of longitudinal subfibrils, indicative of the inner architecture of the fibril.

Contemporaneous anatomic collections and scientific papers from the 19th century school of anatomy of Bologna: preliminary report.

Recently, a strict relationship was demonstrated between scientific pathology reports of the 19th century and a large number of specimens from the museum of pathology 'Cesare Taruffi' of Bologna. Such an experience suggested verifying whether a similar relationship exists between the 19th-century collections of the museum of anatomy and the contemporaneous anatomic scientific literature. The purpose of this preliminary report is to illustrate the first documented samples recovered in Bologna in order to promote such an inventory of old anatomic and pathologic specimens in other museums.

Collagen structure and functional implications.

The bio-mechanical requirements to which the connective tissue is subjected suggest that a causal correlation exist between the substructure and the collagen fibril function. We discuss the relationship between the inner structure of collagen fibrils, their diameter, their spatial layout and the functional requirements they have to withstand, and suggest that collagen fibrils may belong to two different forms indicated as "T-type" and "C-type". The first class, consisting of large, heterogeneous fibrils, parallely tightly packed, subjected to tensile stress along their axis is found in highly tensile structures such as tendons, ligaments and bone. The other class, consisting of small, homogeneous fibrils, helically arranged, resisting multidirectional stresses, is mostly present within highly compliant tissues such as blood vessel walls, skin and nerve sheaths. What causes these architectures to appear is discussed in detail in this review.

A histological and electron-microscopic study of the architecture and ultrastructure of human periodontal tissues.

The structure of periodontal tissues is still far less understood than their clinical relevance would demand. Here the periodontal ligament and radicular cementum in healthy human teeth were studied by light microscopy, transmission and scanning electron microscopy. These observations showed that the extracellular matrix of periodontal ligament is composed of a loose plexus of wavy collagen fibrils immersed in a highly hydrated interfibrillar matrix. Only close to their cemental insertion do these fibrils gather in thick, parallel fascicles (Sharpey's fibres). As these cross the mineralization front, they become infiltrated by the mineral phase and continue directly with the cementum matrix. Sharpey's fibres, "extrinsic" and "intrinsic" fibres all appear to be the same fibres, which bend and branch repeatedly during their course within the thickness of the cementum. Because of its physical continuity with the cementum, a limited portion of the periodontal ligament approximately corresponding to the length of Sharpey's fibres remains unaffected by enzymatic digestion of the interfibrillar matrix while the rest of the ligament is completely dissolved. The findings here indicate that the periodontal ligament and dental cementum join by a continuity rather than a contiguity of structures; that the collagen-mineral relation in cementum has distinctive features in comparison to other hard tissues; that extrinsic and intrinsic fibres of cementum and the adjoining portion of periodontal ligament form a structural, mechanical and metabolic unit distinct from the central, more metabolically active portion of the periodontal ligament.

Different patterns of collagen-proteoglycan interaction: a scanning electron microscopy and atomic force microscopy study.

The extracellular matrix of unfixed, unstained rat corneal stroma, visualized with high-resolution scanning electron microscopy and atomic force microscopy after minimal preliminary treatment, appears composed of straight, parallel, uniform collagen fibrils regularly spaced by a three-dimensional, irregular network of thin, delicate proteoglycan filaments. Rat tail tendon, observed under identical conditions, appears instead made of heterogeneous, closely packed fibrils interwoven with orthogonal proteoglycan filaments. Pre-treatment with cupromeronic blue just thickens the filaments without affecting their spatial layout. Digestion with chondroitinase ABC rids the tendon matrix of all its interconnecting filaments while the corneal stroma architecture remains virtually unaffected, its fibrils always being separated by an evident interfibrillar spacing which is never observed in tendon. Our observations indicate that matrix proteoglycans are responsible for both the highly regular interfibrillar spacing which is distinctive of corneal stroma, and the strong interfibrillar binding observed in tendon. These opposite interaction patterns appear to be distinctive of different proteoglycan species. The molecular details of proteoglycan interactions are still incompletely understood and are the subject of ongoing research.

How to defuse compliance time bombs. Part 2: Six hypothetical allegations. Panel discussion.

In the second and final part of this series, a panel of legal experts discusses more hypothetical allegations against a fictional laboratory, including billing for additional indices, using expired reagents, and falsifying the results of employee competency tests. Learn how to handle these dilemmas before they explode into full-blown legal violations.

Structural and functional macrophages alterations by ceramics of different composition.

Biomaterials may initiate several and complex biological reactions in host tissues, and the cell-biomaterial interactions can determine the release of mediators including monocytes and lymphocytes chemotactic factors. The present work was aimed to investigate in vitro the macrophage biological reactions of a natural apatite obtained by heat treatment at 400 degrees C of bovine bone, compared to other ceramics usually used for dental and orthopedic applications, using synthetic apatite and three types of alumina as controls. Particles chemotactic activity and powders oxidative burst evidenced no monocyte macrophages sensitivity reaction for natural and synthetic hydroxyapatite powders at great granulometry (> 50 microm); data were confirmed by ultrastructural observations; electron microscopy analysis showed macrophages with the features of healthy cells in the presence of both natural and synthetic apatites while macrophages grown in the presence of alumina seemed to be negatively affected. In conclusion, among all ceramics tested, natural apatite displayed a good compatibility with living cells, being better tolerated than synthetic hydroxyapatite which in turn is better tolerated than alumina.

Peri-implant medullary cisternae at the interface of bone-smooth surface titanium endosseous implant.

A histological and ultrastructural study was carried out on the spongy bone response to smooth titanium oral implant surfaces. The samples obtained both from monkeys and from patients at various times from the implant insertion revealed that the bone-implant integration developed through different morphological aspects. The implant surface appeared in contact with medullary lacunae, as well as with osteoid tissue or directly with bone matrix. The complementary ultrastructural techniques employed have shown that the medullary lacunae appeared as wide and flattened cisternae delimited by a continuous single layer of flattened cells forming a thin lamina adhering to the implant and an endosteal lamina facing the bone surface. For their position and flattened shape we named them peri-implant medullary cisternae. The presence of blood vessels, reticular cells and myeloid cells in their lumen suggested that these peri-implant medullary cisternae were functional sites of new bone formation.

Intertwined Sharpey fibers in human acellular cementum.

We had carried out a detailed morphological study on the human acellular extrinsic fiber cementum (AEFC) in order to support the exclusively extrinsic origin of matrix collagen bundles. Mesial and distal cervical third of fresh premolars from young individuals were examined. Semi-thin and thin section clearly show the Sharpey fibres entering in the cementum at right-angle to the root surface and coursing throughout the cementum to the cemento-dentinal junction. On their way to the dentin the Sharpey fibres divide into smaller bundles which, coursing obliquely or tangentially, intersect with others deriving from neighbouring Sharpey fibres. Both de-proteinated and decalcified samples observed at SEM present Sharpey fibres along the fractured surfaces entering and running trough the cementum perpendicularly to the root surface. Fibril bundles are seen branching out from the main body of a single Sharpey fibre and coursing obliquely or perpendicularly to the original fibre. These morphological evidences obtained both at TEM and SEM further confirm that in AEFC fibril bundles running parallel or obliquely to the root surface are branches of Sharpey fibres and not intrinsic cementum fibres.