Qualitative study of young,adult, and aged wistar rats temporomandibular synovial membrane employing light,scanning, and transmission electron microscopy

The aim of this study was to analyze the rat temporomandibular joint (TMJ) synovial membrane at different ages using light, scanning, and transmission electron microscopy. Under light microscopic analysis, the TMJ structures were observed such as condyle, capsule, disk, the synovial membrane collagen type, and cells distribution. In the scanning electron microscopy, the synovial membrane surface exhibited a smooth aspect in young animals and there was an increase with ageing in the number of folds. The transmission electron microscopic analysis showed more synoviocytes in the synovial layer in the young group and still a great number of vesicles and cisterns dilation of rough endoplasmic reticulum in the aged group. In the three groups, a dense layer of collagen fibers in the synovial layer and cytoplasmic extensions were clearly seen. It was possible to conclude that synovial membrane structures in aged group showed alterations contributing to the decrease in joint lubrication and in the sliding between disk and joint surfaces. These characteristic will reflect in biomechanics of chewing, and may cause the TMJ disorders, currently observed in clinical processes. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.     

Structural arrangement of the cardiac collagen fibers of healthy and diabetic dogs

The heart is composed by a specialized muscle, whose form and function are essentials for an adequate work and shows an amount of connective tissue which support and provide insertion for this muscle, whose collagen fibers are responsible for determination of tissue feature. Our objective was to identify the structural arrangement of the heart collagen fibers in dogs. The hearts of the dogs were submitted to the process of the controlled digestion with NaOH solution and observed by scanning electron microscope. Our results showed that the collagen fibers of the endomysial wall have structural arrangement composed by an irregular network with one layer in normal dogs but in diabetic dogs the network acquires a greater amount of the fibers and layers, looking like a "rug" of fibers modifying the relationships of the stress/strain of the tissue. Ahead of the observed results we are able to conclude that exist increase in the amount and thickness of cardiac collagen fibers, beyond the increase of layers and architectural disarrangement in the endomysial wall in the diabetic dogs.     

Biomechanical behavior and histological organization of the three-layered passive esophagus as a function of topography

The zero-stress state of the mucosa-submucosa and two muscle esophageal layers has been delineated, but their multi-axial response has not, because muscle dissection may not leave tubular specimens intact for inflation/extension testing. The histomechanical behavior of the three-layered porcine esophagus was investigated in this study, through light microscopic examination and uniaxial tension, with two-dimensional strain measurement in pairs of orthogonally oriented specimens. The two-dimensional Fung-type strain-energy function described suitably the pseudo-elastic tissue response, affording faithful simulations to our data. Differences in the scleroprotein content and configuration were identified as a function of layer, topography, and orientation, substantiating the macromechanical differences found. In view of the failure and optimized material parameters, the mucosa-submucosa was stronger and stiffer than muscle, associating it with a higher collagen content. A notable topographical distribution was apparent, with data for the abdominal region differentiated from that for the cervical region, owing to the existence of inner muscle with a circumferential arrangement and of outer muscle with a longitudinal arrangement in the former region, and of both muscle layers with oblique arrangement in the latter region, with thoracic esophagus being a transition zone. Tissue from the mucosa-submucosa was stronger and stiffer longitudinally, relating with a preferential collagen reinforcement along that axis, but more extensible in the orthogonal axis.     

Imaging of collagen type III in fluid by atomic force microscopy

Type III collagen is a component of the basement membrane of endothelial cells, and may play a role in the interaction between hemostatic system proteins and the basement membrane of blood vessels. To begin to investigate these structural interactions, we have imaged type III collagen in solution by atomic force microscopy. A 20 microg/ml solution of type III collagen in bicarbonate buffer (pH 9.5) from calf skin was deposited onto a freshly cleaved mica substrate. Atomic force microscopy images were acquired using a fluid cell and tapping mode with oxide-sharpened silicon nitride probes 2, 3, and 4 hours after deposition of the collagen onto the mica. Two-hour preparations displayed fibrillar networks with well-defined sites of nucleation and lateral growth. At 3 and 4 hour polymerizations, more mature fibrils of increasing lengths, diameters, and complexity were observed. Fibrils appeared to be aligning and twisting (helical formation) to form a mature fibril with a higher mass per unit area. Interestingly, the mature fibrils appeared larger centrally with tapered ends displaying declining slopes. These observations compare favorably with those previously published on collagen type I assembly [Gale et al. (1995) Biophys. J. 68:2124-2128]. High resolution atomic force microscopy images of type III collagen in solution should provide a template for observation of the interactions between basement membrane components and hemostatic system proteins present in cardiovascular disease.     

Arrangement and fine structure of collagen fibrils in the decidualized mouse endometrium

The adaptations of the mouse uterus to pregnancy include extensive modifications of the cells and extracellular matrix of the endometrial connective tissue that surround the embryos. Around each implanted embryo this tissue redifferentiates into a transient structure called decidua, which is formed by polygonal cells joined by intercellular junctions. In the mouse, thick collagen fibrils with irregular profile appear in decidualized areas of the endometrium but not in the nondecidualized stroma and interimplantation sites. The fine organization of these thick fibrils has not yet been established. This work was addressed to understand the arrangement and fine structure of collagen fibrils of the decidua of pregnant mice during the periimplantation stage. Major modifications occurred in collagen fibrils that surrounded decidual cells: (1) the fibrils, which were arranged in parallel bundles in nonpregnant animals, became organized as baskets around decidual cells; (2) very thick collagen fibrils with very irregular profiles appeared around decidual cells. Analysis of replicas and serial sections suggests that the thick collagen fibrils form by the lateral aggregation of thinner fibrils to a central fibril resulting in very irregular profile observed in cross sections of thick fibrils. The sum of modifications of the collagen fibrils seem to represent an adaptation of the endometrium to better support the decidual cells while they hold the embryos during the beginning of their development. The deposition of thick collagen fibrils in the decidua may contribute to form a barrier that impedes leukocyte migration within the decidua, preventing immunological rejection of genetically dissimilar embryonic tissues.     

Collagen arrangement in space of Disse correlates with fluid flow in normal and cirrhotic rat livers

Little is known about collagen arrangement in the space of Disse was related to the fluid flow both in normal and cirrhotic liver. We examined the changes in the arrangement of type‐I collagen in thioacetamide‐induced cirrhotic rat livers with immunohistochemistry and SEM after maceration of the noncollagenous tissues with NaOH. The sparse bundles of collagen fibers in the spaces of Disse were mostly elongated fibers with a disorganized arrangement in each nodule. They connected with the broad fibrous septa. Based on a comparison of the architecture of the collagen fibers and the established flow of fluid in the space of Disse, we hypothesize that the fluid in the space of Disse streams along collagen fibers in all directions to broad fibrous septa. The appearance of perinodular plexus in cirrhotic rat livers probably helps to reduce portal hypertension. Microsc. Res. Tech. 78:187–193, 2015. © 2014 Wiley Periodicals, Inc.     

TOC ‐ Issue Information

Knowledge of the collagen structure of an Achilles tendon is critical to comprehend the physiology, biomechanics, homeostasis and remodelling of the tissue. Despite intensive studies, there are still uncertainties regarding the microstructure. The majority of studies have examined the longitudinally arranged collagen fibrils as they are primarily attributed to the principal tensile strength of the tendon. Few studies have considered the structural integrity of the entire three‐dimensional (3D) collagen meshwork, and how the longitudinal collagen fibrils are integrated as a strong unit in a 3D domain to provide the tendons with the essential tensile properties. Using second harmonic generation imaging, a 3D imaging technique was developed and used to study the 3D collagen matrix in the midportion of Achilles tendons without tissue labelling and dehydration. Therefore, the 3D collagen structure is presented in a condition closely representative of the in vivo status. Atomic force microscopy studies have confirmed that second harmonic generation reveals the internal collagen matrix of tendons in 3D at a fibril level. Achilles tendons primarily contain longitudinal collagen fibrils that braid spatially into a dense rope‐like collagen meshwork and are encapsulated or wound tightly by the oblique collagen fibrils emanating from the epitenon region. The arrangement of the collagen fibrils provides the longitudinal fibrils with essential structural integrity and endows the tendon with the unique mechanical function for withstanding tensile stresses. A novel 3D microscopic method has been developed to examine the 3D collagen microstructure of tendons without tissue dehydrating and labelling. The study also provides new knowledge about the collagen microstructure in an Achilles tendon, which enables understanding of the function of the tissue. The knowledge may be important for applying surgical and tissue engineering techniques to tendon reconstruction.     

Morphometric analysis of human sciatic nerve perineurial collagen type IV content

Objectives: Aging is the process which unavoidably alters structure and function of the basal membranes in humans. Though, collagen type IV presents the most prominent component of the basal membranes, we estimated its presence in the perineurium of the human sciatic nerve samples during the aging process. Materials and methods: Material was 12 sciatic nerve samples, obtained from cadavers whose age ranged from 36 to 84 years. Cadavers were classified into three age groups: first which age ranged from 35 to 54 years, second which age ranged from 55 to 74 years and third which included cases older than 75 years. Tissue slices were further stained by labeled streptavidin–biotin method with collagen type IV monoclonal antibody and analyzed with light microscope under 100× lens magnification with oil immersion. Digital images of sciatic nerve perineurium were further processed and analyzed with ImageJ software. Results: Our results showed that there is statistically significant increase of perineurial area, perimeter, collagen type IV area, and collagen type IV area per perineurial perimeter unit in the third age group. These parameters also increased in the second age group, but this increase was not significant. Multiple regression analysis showed that beside fascicular size, age more significantly predict perineurial collagen type IV content. Conclusions: Results of morphometric and statistical analysis pointed to the conclusion that there is significant increase of sciatic nerve perineurial thickness during the aging process. This increase might represent the consequence of perineurial collagen type IV deposition with aging. Microsc. Res. Tech., 2011. © 2011 Wiley Periodicals, Inc.     

Lamellar subcomponents of the cuticular cell membrane complex of mammalian keratin fibres show friction and hardness contrast by AFM

There is a substantial body of information indicating that 18‐methyleicosanoic acid (18‐MEA) is covalently linked to the outer surface of all mammalian keratin fibres and also forms the outer β‐layer of the cuticular cell membrane complex (CCMC) which separates the cuticle cells from each other. Low cohesive forces are expected between the lipid‐containing outer β‐layer and the δ‐layer of the CCMC, thus providing a weak point for cuticular delamination and presenting a fresh layer of 18‐MEA to the newly exposed surface. We have used lateral force microscopy and force modulation atomic force microscopy (AFM) to examine human hair fibres in which the non‐covalently linked fatty acids have been removed. Examination of the lateral force images of new cuticle surfaces revealed by the attrition of overlying cuticle layers showed three separate zones of clearly defined frictional contrast. These are thought to correspond with the δ‐layer, the proteinaceous epicuticle and outer β‐layers of the CCMC. The δ‐layer was found to have a thickness of 16 nm (SD = 1 nm, n = 25), comparable to the 18.0 nm thickness measured from transverse cross‐sections of fibres with transmission electron microscopy. Force modulation AFM showed that the outer β‐layer was softer than the epicuticle and the δ‐layer. The frictional contrast was removed following treatment with methanolic KOH (0.1 mol dm^?3) at 25 °C for 30 min, suggesting the hydrolysis of the thioester linkage and removal of 18‐MEA from the surface.     

The process of collagen biomineralization observed by AFM in a model dual membrane diffusion system

Investigation and simulation of naturally occurring mineralization can offer some new ideas in the design and fabrication of new functional materials for bone analogues. In this paper, a model dual membrane diffusion system (DMDS) was used to study the mineralization behaviour of collagen. The process of mineralization was observed by atomic force microscope (AFM). The results showed that the surface roughness and hardness of mineralized collagen fibers increased with time during the process of mineralization. The adhesion force of mineralized collagen fibers decreased with mineralization time. The micromechanical properties and microstructure changes of mineralized collagen fibers suggested that the mineralization was a step-by-step assembling process.     

Ultrastructure of proteoglycans in the tectorial membrane

The ultrastructure of proteoglycans (PGs) in the tectorial membrane (TM) of the mature chinchilla cochlea was investigated using the cationic dye Cuprolinic blue. When used at a high critical electrolyte concentration, Cuprolinic blue has been shown specifically to bind to the glycosaminoglycan residues of sulfated PGs. After Cuprolinic blue treatment, PGs were observed in the TM which were represented as rod-shaped, electron-dense structures. A perifibrillar, primarily orthogonal, array of PGs was associated with the type A protofibrils. These PGs were distributed in 50 nm intervals along the length of the type A protofibrils. A less common orientation was parallel to the axis of the type A protofibrils. PGs did not appear to be associated with the type B protofibrils. Based upon previous results by other investigators, the TM contains types II and IX collagen, and it appears likely that the type A protofibrils are composed of collagen type II. PGs visualized in the TM in this study thus may represent the glycosaminoglycan residue of type IX collagen which is associated with the type II collagen fibrils. Alternatively, the TM PGs may be small dermatan or chondroitin sulfate PGs.     

Observation of the ultrastructure of anterior cruciate ligament graft by atomic force microscopy

This study presented the fibril ultrastructure of retrieved grafts from the reconstruction of anterior cruciate ligament (ACL) using atomic force microscopy (AFM). The tapping mode images of the AFM were taken from different areas of the longitudinally cut grafts. Regular arrangement of collagen fibrils was found in certain areas of the graft. In many areas, however, the fibrils were not well arranged in a single direction, with some smaller fibrils oriented vertically to larger parallel fibrils. The crossing and tangling of fibrils in ACL grafts was well represented in the three‐dimensional AFM image. This abnormality of graft ultrastructure might indicate the possible alteration of the mechanical environment after ACL reconstruction. This study demonstrated the suitability and importance of ultrastructure observation of ACL grafts by AFM. SCANNING 31: 19–23, 2009. © 2009 Wiley Periodicals, Inc.     

Structure and composition of Unio pictorum shell: arguments for the diversity of the nacroprismatic arrangement in molluscs

Mollusc shells are complex organomineral structures, the arrangement and composition depending on the species. Most studies are dedicated to shells composed of an aragonite nacreous and a calcite prismatic layer, so the nacreous prismatic model based on Pinctada and Atrina–Pinna. Here, we studied the micro‐ and nanostructure, the mineralogy and composition of a nacroprismatic bivalve species: Unio pictorum. The prismatic layer of Unio is aragonite, and the inner structure of the prismatic units strongly differs from those of the calcitic layers. The shape of the prisms varies depending on their growth stage. The first layers of nacre are similar to those of gastropods (columnar nacre), then evolve towards the typical bivalve arrangement (sheet nacre). Na, Sr, Mg, P and S are present in both prisms and nacre. The organic prismatic envelopes are rich in sulphur amino acids, whereas organic sulphate is present within the prisms and the nacreous tablets. P is present as phosphate, probably a mixture of organic and mineral complex. Chemical distribution maps confirm the absence of an organic membrane between the nacre and the prisms. The comparison of the structure, mineralogy and composition of Unio pictorum and different species show the diversity of nacroprismatic shells, and that these features are taxonomically dependent.     

Comparative ultrastructure of the fertilized egg envelope in Corydoras adolfoi and Corydoras sterbai,Callichthyidae, Teleostei

In teleost, the structural characteristics of fertilized egg and egg envelope are very important for classification of genus or species. The structures of fertilized egg and egg envelope from Corydoras adolfoi and Corydoras sterbai, Callichthyidae, Siluriformes in teleost were examined by scanning and transmission electron microscopes to confirm whether these morphological structures have specificities of species and family or not. The fertilized eggs of C. adolfoi and C. sterbai were non‐transparent, spherical, demersal, and strong adhesive. There were no structural differences between two species through the light microscope. The size of the fertilized eggs of C. adolfoi was 1.95 ± 0.03 mm (n = 20), and that of C. sterbai was 1.92 ± 0.03 mm (n = 20). The perivitelline space was almost not developed in both species. In both species, the adhesive protuberances structures were on the outer surface of egg envelope. And fibrous structures were specially located at attachment part of spawning bed. And the egg envelope consisted of two layers, an inner lamellae layer and an outer strong adhesive layer with high electron dense protuberances structures in cross section. Consequentially, the fertilized eggs, outer surface on the egg envelope and cross section of egg envelope have identical structure. So, these structural characteristics of fertilized eggs and egg envelope show genus Corydoras specificity.     

Freeze-substitution of rabbit tibial articular cartilage reveals that radial zone collagen fibres are tubules

Investigations of the micromorphology of rabbit tibial articular cartilage using scanning and transmission electron microscopy revealed that the collagenous elements in the tissue form fluid-containing tubular structures. The commonly described radial or deep zone longitudinal fibres were found to be tubular structures with internal diameters of 1–2 μm. The walls of the tubules were composed of tightly packed fibrils of collagen. The tangential zone, close to the tibial plateau, was composed mainly of a spongy arrangement of collagen fibrils, containing bunches of tangentially lying small (< 1 μm) diameter tubules. The application of conventional chemical fixation techniques resulted in the fine detail of this tissue being obscured. When the tissue was frozen, followed by cryo-scanning electron microscopy or freeze-drying, prior to observation in the scanning electron microscope the tubule structures were not obviously present. It was only by applying freeze-substitution techniques, followed by critical point drying or resin embedding, that the structure was revealed clearly. Segregation of water into ice crystals did occur during the freezing process, but the formation of those crystals played no part in creating the tubular morphology observed. A similar structure was still revealed following pre-treatment with glycerol, methanol or Triton X-100, provided that concentration of these additives was not too high. The walls of the tubules in the radial region were composed of straight, longitudinally arranged as well as helically arranged, 30 nm diameter fibrils. The lumen of the tubules appears to be lined by a circumferentially arranged array of approximately 10 nm diameter fibres, spaced at regular intervals of 50–70 nm.     

Intercellular junctions in embryonic chick cardiac muscle revealed by rapid freezing and freeze-substitution.

Using the method of rapid freezing and freeze-substitution, the embryonic chick cardiac muscle was investigated by transmission electron microscopy. Initially, the intercellular junctional complexes (fasciae adherentes and desmosomes) were formed in close proximity to each other along a nearly straight line. Subsequently, the separation of fasciae from desmosomes took place to form intercalated discs. The cell membranes of fasciae adherentes were reinforced with highly interwoven fine fibrils at which myofibrils terminated. The intercellular space of fasciae was bridged with fine fibrillar structures seemingly connected by a thin line at their middle portions. In the intercellular space of desmosomes, central lamina and traversing filaments were clearly observed. The outer and inner leaflets of the desmosomal plasmalemma were asymmetrically differentiated; the outer leaflet was thinner than the inner leaflet. On the inner side of the cell membrane, an electron-lucent layer and a dense desmosomal plaque were observed. The latter structure had protrusions with less electron density towards the cytoplasmic side. Further inside, a meshwork of fine fibrils was seen along and toward which bundles of intermediate filaments ran. The results obtained with freeze-substitution appeared to provide more information than those with thin sections after conventional fixation or with replicas of chemically fixed/glycerinated or physically fixed/deep-etched materials.     

Molecular and chemical investigations and comparisons of biomaterials for ocular surface regeneration

This study investigated and compared the ultrastructural and chemical properties of representative biomaterials for ocular surface regeneration: a human amniotic membrane (AM) in a basal plate, a human AM in reflected chorion, a preserved AM, and a human corneo‐scleral tissue. Assessments of the morphological differences in the extracellular matrices were evaluated by hematoxylin–eosin, Masson's trichrome (for total collagen), and picrosirius‐red (for newly synthesized collagen) staining. Assessments of the changes in the molecular structures and chemical compositions of the biomaterials for ocular surface regeneration were evaluated by Raman spectroscopy. A placental AM (52 %) was a dense and thick collagenous structure compared to a reflected AM (23 %). The spectroscopy did not obtain any structural information for a preserved AM. The cornea group (100 %, control) and sclera group (104 %) showed the collagen lamellae and interfibrillar spacing, and a slight inflammatory reaction with more fibrous and granulomatous tissues. There was a formation of newly synthesized collagen in a placental AM, while there were few collagen components in a reflected AM. Human AM tissues showed consistent Raman spectra and the characteristic collagen bands, similar to the corneal and scleral tissues. Therefore, these findings suggest that human placental AM and reflected AM are structurally suitable for scleral and corneal surface regeneration, respectively, while human placental or preserved AM and reflected AM are molecularly and chemically suitable for corneal and scleral surface regeneration, respectively. Microsc. Res. Tech. 77:183–188, 2014. © 2013 Wiley Periodicals, Inc.     

Collagen types I, III, and V constitute the thick collagen fibrils of the mouse decidua

A mammal's endometrium is deeply remodeled while receiving and implanting an embryo. In addition to cell proliferation and growth, endometrial remodeling also comprises synthesis and degradation of several molecular components of the extracellular matrix. All of these events are orchestrated by a precise sequence of ovarian hormones and influenced by several types of cytokines. As we have previously reported, an intriguing and rapid increase in collagen fibril diameter occurs in the decidualized areas of the endometrium, surrounding the implantation crypt, whereas collagen fibrils situated far from the embryo remain unchanged. Collagen fibrilogenesis is a complex molecular process coordinated by a number of factors, such as the types and amounts of glycosaminoglycans and proteoglycans associated with collagen molecules. Collagen genetic type, mechanical stress, aging, and other factors not yet identified also contribute to this development. A recent study suggests that thick fibrils from mouse decidua are formed, at least in part, by aggregation of thin fibrils existing in the stroma before the onset of decidualization. In the present ultrastructural study using single and double immunogold localization, we showed that both thin and thick collagen fibrils present in the mouse pregnant endometrium endometrium are heterotypic structures formed at least by type I, type III, and type V collagens. However, type V collagen predominates in the thick collagen fibrils, whereas it is almost absent of the thin collagen fibrils. The putative role of type V homotrimer in the rapid increase of the diameter of collagen fibrils of the mouse decidua is discussed.     

Irregular Shaped,Assumably Semi‐Crystalline Calciumphosphate Platelet Deposition at the Mineralization Front of Rabbit Femur Osteotomy: A HR‐TEM Study

Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high‐resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow‐up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex‐shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix. SCANNING 35: 169‐182, 2013. © 2012 Wiley Periodicals, Inc.