Distribution and organization of the elastic system fibres in healthy human gingiva

Summary The ultrastructural distribution and organization of the elastic system fibres, i.e. oxytalan, elaunin and elastic fibres, were studied by transmission electron microscopy and by an immunohistochemical method for the detection of elastin in healthy human gingiva. The morphological distribution of these fibres was characterized by the presence of oxytalan, elaunin and elastic fibres, respectively, in the upper, medium, and deep layers of gingival connective tissue. Anti-elastin antibody reacted with microfibrils and amorphous material of the elastic system fibres throughout the gingival connective tissue. These findings were interpreted as indicating that the microfibrils were associated with small amounts of elastin at their surface.     

Structure and Development of the Notochord ‘Elastica Externa’ and Nearby Components of the Elastic Fibre System of Agnathans

Between 15 days and 3 months in age, the ‘elastica externa’ of the notochord sheath of larval lampreys develops from patches of moderately dense and amorphous material into a thick, continuous and electron-dense layer. In both lampreys and hagfish, this layer stains strongly with Verhoeff's elastic stain and aldehyde fuchsin and is penetrated by collagen fibrils on both its outer and inner boundaries. Peroxidase labelling using an antibody raised against human elastin specifically labels both the notochord ‘elastica externa’ and the elastic fibre system of lampreys. The diameters of the microfibrils (10–13 nm) of the oxytalan, elaunin and elastic fibres of lampreys and hagfish are the same as those of higher vertebrates. The connective tissue immediately dorsal and ventral to the notochord of lampreys contains mainly oxytalan fibres in very young ammocoetes, a combination of oxytalan, elaunin and elastic fibres in older ammocoetes, and predominantly elastic fibres in adult lampreys. While the region of the endomeninx at the base of the spinal cord contains almost exclusively oxytalan fibres in young ammocoetes, it also possesses numerous elastic fibres in adult lampreys. These findings indicate that, as in higher vertebrates, the elastic fibres of lampreys develop from oxytalan fibres via elaunin fibres.     

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.     

Electron microscopic study on the elastic and elastic related fibres in the human fascia transversalis at different ages

An electron microscopic study of the elastic fibre and elastic related fibres of the fascia transversalis of the human inguinal triangle was performed in 20 male patients aged 13 to 81 a with right indirect inguinal hernia submitted to surgical repair. The 3 fibre types comprising the elastic system (oxytalan, elaunin, and elastic fibres) tend to be ordered in a precise manner and sequence among the fibrils, fibres, and collagen fibre bundles, respectively. The present findings show that with aging, there is a decrease in the oxytalan fibres and an increase in the amorphous substance of the elastic fibres. The authors concluded that the decrease in oxytalan fibres as a function of age may be responsible for alteration in the resistance of the transversalis fascia.     

Peptide stimulation of phagocytosis in Amoeba proteus

Summary Normal articular cartilages from the weightbearing areas of the femoral condyles of the knee joints of 11 patients (3–20 years old) and of 35 Schwarzkopf sheep (3 months to 2 years old) were studied using the electron microscope. The study has shown that the matrix of normal articular cartilage is not only composed of collagen fibrils and proteoglycans, but also contains two types of elastic system fibres. Small elastic fibres can be identified in the superficial and lower radiate zones of cartilage of man and sheep. Similar to elastic fibres in other tissues, they consist of a central amorphous core and are surrounded by aggregates of 10 nm microfibrils. Another type of elastic system fibres, oxytalan fibres, are found in the intermediate and upper radiate zones of the articular cartilage.     

Immunohistochemical characterization of elastic system fibers in rat molar periodontal ligament.

Among elastic system fibers, oxytalan fibers are known as a ubiquitous component of the periodontal ligament, but the localization and role of elastin-containing fibers, i.e., elastic and elaunin fibers, has yet to be clarified. In this study, we immunohistochemically investigated the localization of elastin and fibrillin, major proteins of elastin-containing fibers in the periodontal ligament of rat lower first molars. At the light microscope level, distribution of elastin-positive fibers was not uniform but often concentrated in the vicinity of blood vessels in the apical region of the ligament. In contrast, fibrillin-positive fibers were more widely distributed throughout the ligament, and the pattern of their distribution was comparable to the reported distribution of oxytalan fibers. At the ultrastructural level, assemblies or bundles of abundant fibrillin-containing microfibrils were intermingled with a small amount of elastin. This observation indicated that elastin-positive fibers observed under the light microscope were elaunin fibers. No mature elastic fibers, however, were found in the ligament. These results show that the major components of elastic system fibers in the periodontal ligament of the rat mandibular first molar were oxytalan and elaunin fibers, suggesting that the elastic system fibers play a role in the mechanical protection of the vascular system.     

Ultrastructure and staining properties of aortic microfibrils (oxytalan)

Microfibrils are the insoluble, 10- to 12-nm components of the extracellular matrix that are involved in elastogenesis. Reports of their ultrastructure vary: they have been described as tubular and beaded and as nontubular filaments that are devoid of any periodicity. Ultrastructurally, microfibrils resemble oxytalan fibers that have been observed in peridontal membranes, skin, and other locations. Whether microfibrils have the staining characteristics of oxytalan is difficult to determine in tissues because available light microscopic stains also stain elastin. Calf aortic smooth muscle cells grown in media without added ascorbate provide a unique model for examining the ultrastructure and staining characteristics of chemically defined microfibrils. Microfibrils are the predominant insoluble extracellular protein in such cultures, which do not deposit collagen or elastin. These studies demonstrate that microfibrils are tubular structures with 10- and 12-nm striations and have the same staining characteristics as oxytalan, reacting with aldehyde fuchsin and orcein after oxidation. Microfibrillar protein is enriched in glutamic and aspartic acids and the electron density of microfibrils is enhanced by fixation in the presence of cationic dyes. In such preparation, microfibrils are made visible within the core of amorphous elastin as well as in regions that are free of elastin. The widespread distribution of microfibrils (oxytalan) indicates that their function extends beyond elastogenesis. Their localization within tissues suggests that they serve as an elastic attachment protein in sites that are subject to mechanical stress.     

Ultrastructural cytochemical properties of elastinassociated microfibrils and their relation to fibronectin

Summary The characteristics of elastin-associated microfibrils were investigated in the tunica adventitia of mouse aortas at the ultrastructural cytochemical level. The high iron diamine-thiocarbohydrazide-silver proteinate (HID-TCH-SP) method specific for sulphate groups was used with and without prior treatment ofen bloc specimens with either monopersulphate or cupric sulphite reagent. Amorphous elastin formed a clearly identifiable central core with microfibrils located both peripherally and interstitially. Sequential oxidation with monopersulphate and HID-TCH-SP demonstrated a characteristic staining for oxytalan fibres and intensely stained the microfibrils, whereas amorphous elastin stained weakly. Sequential thiosulphation with cupric sulphite and HID-TCH-SP for the demonstration of disulphide linkages and sulphydryl groups intensely stained microfibrils and weakly to moderately stained the amorphous elastin. This reactivity of the microfibrils was not altered by digestion with chondroitinase ABC, performed prior to or after treatment with either monopersulphate or cupric sulphite. In the specimens not exposed to either monopersulphate or cupric sulphite there was no definite HID-TCH-SP staining of microfibrils and amorphous elastin. Further, immunostaining with rabbit antibody specific for mouse fibronectin localized fibronectin in the microfibrils but not in the amorphous, elastin. These results indicate that elastin-associated microfibrils in mouse aorta lack stainable sulphate complex carbohydrates but are enriched with either disulphide or sulphydryl groups, or both, and further demonstrate the close correlation between these glycoproteins and fibronectin.     

Ultrastructural visualization of elastic fibres with a tannate-metal salt method

Summary A modification of the tannic acid-metal salt method was applied as an ultrastructural stain for elastin. Thin sections of glutaraldehyde-fixed, embedded rat aorta and rabbit elastic cartilage, with and without osmication, were examined. Raising the pH of the tannic acid solution from 2.7 to 9.0 progressively increased the electron-density of elastic fibres and collagen fibrils in osmicated and unosmicated specimens. The maximum tannic acid staining of elastic fibres was observed in the pH range 7.0–9.0. Collagen staining, although less intense than that of elastic fibres, was also greatest in this pH range. Elastic fibres in osmicated specimens demonstrated the strongest tannic acid staining with a minimal increase in density of collagen and cell nuclei when compared to the unosmicated specimens. Sequential treatments of osmicated specimens with tannic acid pH 7.0–9.0, and uranyl acetate, pH 4.1, enhanced the density of the elastin intensely, increased collagen staining moderately, but hardly increased the density of nuclei and microfibrils. In elastase-digested osmicated specimens, all tannic acid (pH 7.0)-uranyl acetate-reactive elastin was selectively removed. These results demonstrate that all the neutral and alkaline tannic acid-uranyl acetate methods can be used as a postembedment stain for elastin specimens fixed in glutaraldehyde and osmium tetroxide.     

Subepithelial elastic system fibers of oblique mucosal folds in the rat proximal colon.

The proximal colon of the rat is characterized by a 'herring bone' pattern of oblique mucosal folds (OMF) which are arranged in a parallel array. By light and electron microscopy the OMF exhibited rich subepithelial elastic system fibers which bound the epithelial basement membrane and the smooth muscle cells of the lamina propria together. The elastic system fibers usually consist of elastic, elaunin and oxytalan fibers. However, the subepithelial elastic system fibers of the OMF were composed of relatively thin elastic fibers with a few microfibrils, and elaunin and oxytalan fibers which were almost indiscernible. Areas other than the OMF were quite poor in subepithelial elastic system fibers. The interpositions between each of the OMF were composed of typical components: elastic, elaunin and oxytalan fibers. The composition of the subepithelial elastic system fibers of the OMF does not correspond to that of any other organs previously reported. The present study suggests that the OMF of the rat proximal colon might be equipped in such a way to resist to distension or compression.     

The Use of Tannic Acid-Glutaraldehyde in the Study of Elastic and Elastic-Related Fibers

Fixation with tannic acid—glutaraldehyde permits distinction of oxytalan, elaunin and elastic fibers in the electron microscope. The results obtained using tannic acid at concentrations of 1.0%, 0.5% and 0.25% in 3% glutaraldehyde were compared. The 0.25% concentration is recommended for studying fine details of connective fibrils and for regular staining of elastin.     

Fine mapping of tropoelastin-derived components in the aorta of developing chick embryo

Summary Affinity-purified antitropoelastin antibodies have been used to localize tropoelastin-derived components in aortas from chick embryos of different age by immunoelectron microscopy. Staining in the matrix is first noted at day 3 associated with irregular bundles of filaments resembling microfibrils, in the absence of amorphous elastin deposits. Amorphous material, which rapidly accumulates at later stages, is heavily labelled, while surrounding microfibrils are only poorly labelled. By contrast, a more intense staining of microfibrils persists in regions in which amorphous material is not morphologically evident. These observations indicate that the initial accumulation of elastin requires microfibrils, while the two components are not in close association in the subsequent growth of the amorphous core of the fibre. Intracellular staining is evident in the secretory apparatus of the cell and in peripheral large vesicles. Differentiated cells also show regions of close contact with elastic fibres in which immunological staining for elastin is very close to the cell membrane.     

Ultrastructural cytochemistry of oxytalan fibres in monkey periodontal ligaments with the high iron diamine method

Summary Monkey periodontal ligaments have been examined at the ultrastructural level to demonstrate the nature of reactive sites in oxytalan fibres. The high iron diamine (HID) and HID-thiocarbohydrazide-silver proteinate methods specific for sulphate groups, with and without prior oxidation with monopersulphate, were used. Oxytalan fibres were composed of bundles of microfibrils with a diameter of 11.5 ± 1.7 nm (mean ±s.d.,n = 50). In cross section the microfibrils were found to have a denser periphery, giving them a tubular appearance. The oxytalan microfibrils of non-oxidized specimens showed little reactivity with either HID method, except that the extracellular matrix material in close association with collagen fibrils stained weakly; in oxidized specimens, both HID methods strongly stained oxytalan microfibrils and weakly stained the extracellular matrix material. Such reactivity of oxytalan microfibrils was not altered by digestion with testicular hyaluronidase or chondroitinase ABC, performed prior to or after persulphate oxidation. Further, the sequential thiosulphation and HID method for the demonstration of disulphide and sulphhydryl groups stained oxytalan fibres moderately. These results indicate that the oxidative generation of sulphate groups in oxytalan fibres may occur from either disulphide or sulphhydryl groups, or both, rather than the result of unmasking of sulphated glycosaminoglycans.     

The elastic fibre system in the veins of the human lower oesophagus

The elastic, elaunin and oxytalan fibres in the tunica mucosa and tela submucosa of veins of the human lower oesophagus were studied in 30 necropsy and biopsy specimens using appropriate histological and ultrastructural methodologies. Elaunin fibres predominate in the veins endowed with muscle cells. Scattered oxytalan and elastic fibres were also observed, the latter being more numerous at the vein periphery. No noteworthy fibre arrangement was encountered in veins lacking muscle cells. Those fibres disposed around the veins were considered to belong to the connective tissue of the tunica mucosa and tela submucosa in which they are embedded. The veins of the lower oesophagus seem to be of low elasticity, which may be related to a blockage mechanism described for the gastro-oesophageal blood stream in cases of portal hypertension.     

Microfibrils: a constitutive component of reticular fibers in the mouse lymph node

Summary Fibrous components other than collagen fibrils in the reticular fiber of mouse lymph node were studied by electron microscopy. Bundles of microfibrils not associated by elastin and single microfibrils dispersed among collagen fibrils were present. The diameter of the microfibrils was 13.29±2.43 nm (n=100). Elastin-associated microfibrils occurred at the periphery of the reticular fiber. Elastin was enclosed by microfibrils, thus forming the elastic fiber, which was clearly demonstrated by tannic acid-uranyl acetate staining. In the reticular fiber of lymph nodes, the elastic fiber consisted of many more microfibrils and a small amount of elastin. These microfibrils, together with the collagen fibrils, may contribut to the various functions of the reticular fibers.     

The infrastructure of aortic elastic fibers

Elastic fibers are composed of a central core of elastin that is amorphous and electron-lucent in conventional transmission electron micrographs and peripheral microfibrils. A complex infrastructure within the amorphous elastin of mature rat aorta is made visible by fixation and staining with a glutaraldehyde-ruthenium red mixture in phosphate buffer or osmium-ruthenium red in cacodylate buffer. The infrastructure is composed of at least two interlacing but distinct elastic structural components; a framework of circumferentially orientated microfibrils and a three-dimensional meshwork of filaments that permeate the fiber. The latter resembles a reticulum that has previously been observed in freeze-fractured and negatively stained elastin and attributed to the supramolecular organization of elastin. Microfibrils also extend from the core of the elastic fiber into the surrounding matrix where they appear to function as anchoring fibers. These observations indicate that the elastic properties of the arterial wall are an integrated function of both elastin and microfibrils.     

Particular structure of the anterior third of the human true vocal cord.

The histological aspects of the true vocal cord mucosa change in the anterior third compared with the posterior two thirds. The anterior third is characterized by an epithelium where the ridges, marked in the posterior two thirds, are very slight or even absent. The underlying basement membrane, which is thin in the posterior two thirds, here appears particularly thick. At the ultrastructural level in this area, beneath a normally thickened basal lamina, a thick layer of finely granulated electron-dense material, interspersed with thin and randomly scattered collagen fibrils and proteoglycan filaments, is detectable. Beneath this thickened basement membrane, a layer of small undulated collagen fibril bundles with very numerous interspersed oxytalan fibres is found. The collagen fibrils, small in diameter (30-40 nm), seem to continue with the collagen fibrils of the basement membrane. In this layer numerous blood vessels with a very thick, delaminated basement membrane are also observed. The underlying area is characterized by the vocal cord ligament, composed by large compact collagen fibril bundles with interspersed elastic fibres. The particular features of the thick basement membrane, the thick-walled and delaminated vessels and the modular distribution of the elastic system together may well form the basic structure enabling the functional integration of the vocal ligament into the overlying mucosa and the underlying vocal muscle.     

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.     

THE FINE STRUCTURE OF ELASTIC FIBERS

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.