The collagenic structure of human digital skin seen by scanning electron microscopy after Ohtani maceration technique.

We performed a morphological scanning electron microscope (SEM) study to describe the fine structure and disposition of collagenous tissue in the human toe. After therapeutic amputation of a human right Leg, we applied the Othani maceration technique to the skin of three toes surgically explanted from the foot. We distinguished eight cutaneous regions and focused on some specialized collagenous structures differing in the thickness of the skin. The eight areas investigated were: the dorsal skin, the eponychium, the perionychium, the hyponychium, the region under the visible nail, the nail root, the plantar skin and finally the toe tip. Each of these areas is characterized by a distinctive collagenous surface disposition, with some peculiar features mostly related to dermal. papillae. At high magnification, we observed the spatial arrangement of the cottagen fibers constituting the top of the dermal, papillae that represents the attachment site of the proliferative basal layer of the epidermis. We also noted an impressive density of collagen fibers throughout the thickness of the dermal layer, organized in specialized structures and constituting the skeleton of dermal, thermoreceptorial corpuscles or sweat glands. A combination of SEM and Ohtani technique disclosed the three-dimensional architecture of the collagenous matrix of tarsal skin under physiologic conditions, giving a detailed description of the most reactive tissue during pathologic processes

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 fibrillar crimps of the sclera

Crimps are a typical feature of tendons and of some ligaments where they are responsible for the non-linear behaviour of the tissue at low strain values. Previous studies (Raspanti et al., 2005) shown that in tendons these visible crimps always correspond to a distinctive buckling and/or left-handed torsion of the collagen fibrils, subsequently named “fibrillar crimp” (Franchi et al, 2007). These planar crimps are exclusive of the collagen fibrils of tendons and ligaments, whose subfibrils run straight and parallel and tend to behave like stiff rods; by contrast, the collagen fibrils of most tissues, which are sometimes defined as “reticular fibrils” of “type III fibrils” and whose subfibrils follow an helical course, are almost infinitely flexible and can withstand even sharp U-turns without buckling. In the present study we investigated the rat sclera, whose ultrastructure is closely related to that of tendons and ligaments (Raspanti et al., 1992). The tissue was observed by light microscopy, high-resolution scanning electron microscopy and atomic force microscopy. The sclera appears made of flattened fascicles of large and heterogeneous collagen fibrils, running in all directions and often following a wavy course. The individual fascicles are identical in structure and appearance to those of tendon, as are the numerous crimps which can be easily observed along their fibrils. The presence of crimps in the sclera cannot be taken for granted because at variance with tendon, which is subject to intermittent load/recoil cycles, this tissue is held under continuous tension by the intraocular pressure (IOP). A typical intra-ocular pressure of 20-30 mmHg would induce in the rat sclera a tension varying from 50 to 80 kN/m2, and the elastic behaviour of the crimps may have a role in maintaining the shape of the eyeball. The crimps may therefore be fully functional, confirming this tissue as a sort of hemispherical tendon

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

Ultrastructural aspects of mineralization-induced modifications in turkey tendon

In all tendons the collagen fascicles follow a wavy course (actually a flattened lefthanded helix) forming visible crimps. Each crimp corresponds to a sharp bend and/ or an axial twisting of individual collagen fibrils (Raspanti et al., 2005; Franchi et al., 2010), and even once the fibril are straightened out a permanent local deformation remains visible, still revealing the original crimp location (Raspanti et al., 2005). The tendons of some birds represent a special case as they undergo a physiological process of gradual mineralization involving heavy modifications of the tissue architecture. In the present research, turkey tendons appeared to be more finely subdivided into thinner fascicles than most tendons; they contained a greater amount of cell-rich endotenon tissue as well as occasional nodules of cartilage-like matrix. The most striking 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 mineralized fibrils ran perfectly straight, but the electron microscopy revealed traces of pre-existing crimps locked in the extended position by the mineralization process. The inorganic phase itself appeared composed of two different types of fine particles, respectively growing inside or around the collagen fibrils and looking as tightly packed fine needles or as larger platelets regularly arranged in relation with the D-period. 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

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

3D imaging of the osteon lacunar-canalicular system: an improved corrosion-casting method

The osteocyte lacunar-canalicular system has been the subject of several studies either by serial sectioning of embedded specimens, or by confocal laser scanning microscopy, or with FIB/SEM imaging. With all these techniques the persistence of the bone matrix hampers the visualization of the finer details of the canalicular network. In the present research the methyl-metacrylate casting technique was applied to human cortical bone in order to evidentiate the 3-D organization of the osteons lacunar-canalicular system. The MMA monomer infiltration into the vascular canals, and hence into the lacunar-canalicular system was driven by capillarity, helped by evaporation and the resulting negative pressure. The resin penetration was complete in the Haversian canal but limited to a depth of 4 – 5 layers of lacunae around the canal itself, suggesting that each secondary osteon is a close system limited by an impenetrable cementing line. The casts duplicated the shape, position and connections of the lacunae, and allowed an unrestricted exploration of lacunar-canalicular network without manipulations such as cutting or sawing. Two systems of canalicula could be distinguished: the equatorial, which connects osteocytes lying on the same concentrical level, and the radial, which interconnects different levels. The equatorial canalicula radiate from the lacunar border on a planar surface around the lacuna, forming straight and basically parallel canals; the radial have a predominant direction perpendicular to the equatorial plane. The mean length of the radial canalicula was 38.4 +/- 7.35 um in human osteons, while their mean diameter was 195.7 +/- 79.58 nm. The equatorial canalicula had an average diameter of 249.7 +/- 73.78 nm, significantly larger (p < 0.001) than the radial. The architecture of lacunar-canalicular network obtained by the MMA casts was compared with theoretical models. Our data suggests that the intracanalicular pericellular fluid is static and that the supply of ions, oxygen and metabolites to the osteocytes can be assured by the diffusion of these molecules into the static volume of the solution without the need to hypothesize a fluid flow within the canalicular system