A comparative light microscopic evaluation of oxytalan fiber staining with a variety of dye substances.

Staining of oxytalan fibers in marsupial, eutherian and human periodontal ligaments was surveyed with 65 different dyes. Using the criteria of response to preoxidation, distribution, and morphologic appearance, 27 dye preparations in addition to the Gomori aldehyde-fuchsin, Taenzer-Unna orcein, and Weigert resorcin-fuchsin techniques displayed oxytalan fibers. With two exceptions all dyes were cationic and reacted with varying degrees of excellence with different animals. Most dyes produced their best staining results as concentrated solutions in 3% acetic acid, suggesting involvement of oxidatively engendered polyanions predominantly associated with an acid mucopolysaccharide component of the oxytalan fiber. The significance of carboxyl and sulfur-containing groups should not be overlooked in further studies aiming to elucidate oxytalan fiber chemistry and microstructure. This study supported the view that oxytalan fibers belong to the family of elastic tissues and represent a biologically important system within the periodontal ligament.     

Oxytalan fibers in the periodontal ligament of the Caiman and the Alligator (Crocodilia, Reptilia)

Histological examination of the fibrous and cellular connective tissue components of the periodontal ligament in the Caiman and the Alligator reveals the presence of reticular fibrillae, collagenic, elastic, and oxytalan fibers, as well as fibrocytes, osteoblasts, cementoblasts and epithelial rests. The oxytalan fibers differentiated by the peracetic acid aldehyde-fuchsin method are most numerous in the coronal region, radiating from the primary cementum into the periodontal ligament a short distance. Oxytalan fibers in fewer numbers are found interspersed between the oblique and the horizontal principal fiber bundles. Inasmuch as the crocodilian teeth have continuous replacement and thus a relatively short functional life, the oxytalan fibers of the Caiman and the Alligator appear to be proportionally fewer in number than they are in the mammalian periodontal tissues. The presence of the oxytalan fibers and epithelial rests in the Order Crocodilia (Crocodilia) adds to the number of dental structures shared with the Class Mammalia (Mammalia) (mammals) such as a stellate reticulum, a primary and secondary cementum and a periodontal ligament. This furnishes additional histological evidence for evaluation of the phylogenetic position of this group.     

Ultrastructural localization of fibrillin-1 and fibrillin-2 in oxytalan fibers in periodontal ligament of Japanese Macaca fuscata monkey

Although oxytalan fibers are a ubiquitous component of the periodontal ligament, their composition and function remain to be fully clarified. The purpose of this study was to investigate the localization of fibrillin-1 and fibrillin-2, large cysteine-rich glycoproteins, in monkey molar periodontal ligament by means of immunohistochemistry and electron microscopic immunogold labeling. Fibers positive for both fibrillin-1 and -2 were observed throughout the periodontal ligament, and their pattern of distribution was basically the same as that of oxytalan fibers. Double immunogold electron microscopy revealed that fibrillin-1 co-localized with fibrillin-2 on the microfibrils of the oxytalan fibers. The results suggest that oxytalan fibers in monkey periodontal ligament are made up of fibrillin-rich microfibrils. These results should contribute to a better understanding of the role of oxytalan fibers in development and tissue remodeling in periodontal ligament.     

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.     

Oxytalan fibre organization in marsupial mandibular periodontal tissues

The arrangement and distribution of oxytalan fibers in Australian marsupials has not previously been reported. Periodontal tissues of wombat, wallaby, possum, and marsupial mouse were examined to ascertain oxytalan fibre organization. Despite adaptation of the marsupial masticatory apparatus to different diets the oxytalan fibre organization in the periodontal ligament shows a basic pattern which corresponds with that reported in other animals. The oxytalan system forms a continuous meshwork of fine, branching fibres which completely invests each tooth root and connects adjacent teeth. Thick ribbon-like apico-occlusally orientated oxytalan fibres, thought to form by the coalescence of thinner fibres, are restricted to the periodontal ligament. The oxytalan fibres are embedded in cementum and attached to blood vessels in the periodontal ligament. Oxytalan fibres do not insert into alveolar bone. Histological evidence indicates functional remodelling of the oxytalan fibre system in continuously erupting teeth.     

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.     

The cytology of submandibular glands of the opossum

Tooth attachment in the majority of the bony fish is by ankylosis or fibrous membrane. However, in one group of the osteichthys, the trigger-fish or balistids, tooth attachment is by means of a periodontium composed of a shallow alveolar socket, a periodontal ligament and acellular cementum. Histologically, the balistid periodontal ligament is composed of a dense fibro-cellular connective tissue possessing an abundance of typical fibrocytes, collagen fiber bundles, and oxytalan fibers. The collagen fiber bundles which resemble the principal fiber bundles of the mammalian periodontal ligament are inserted into the bone of the shallow alveolar sockets and are anchored to the teeth by means of a layer of amorphous acellular cementum that covers the radicular dentin. No cementoblasts were found in functional teeth, and epithelial rests are lacking. The mid-central zone of the balistid periodontal ligament is occupied by small blood vessels.     

The fine structure of the oxytalan fiber

Maraglas-embedded sections of periodontal membranes around continually growing incisors of Sprague-Dawley rats fixed consecutively in cold glutaraldehyde and Palade's 1 % buffered osmium tetroxide were examined under the electron microscope for oxytalan connective tissue fibers. Oxytalan fibers were noted to consist of bundles of filaments approximately 150 A in diameter with an interfilamentous amorphous substance of approximately the same diameter. A periodicity of fibrillar elements was not obvious.     

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.     

Comparison of rat and mouse pulmonary tissue mechanical properties and histology.

The present study compares the dynamic mechanical properties and the contents of collagen and elastic fibers (oxytalan + elaunin + fully developed elastic fibers) of mice and rat lung strips. Resistance, elastance (E), and hysteresivity (eta) were obtained during sinusoidal oscillations. The relative amounts of blood vessel, bronchial, and alveolar walls, as well as the mean alveolar diameter were determined. In both species, resistance had a negative and E a positive dependence on frequency, whereas eta remained unchanged. Mice showed higher E and lower eta than rats. Although collagen and elastic fiber contents were similar in both groups, mice had more oxytalan and less elaunin and fully developed elastic fibers than rats. Rats showed less alveolar and more blood vessel walls and higher mean alveolar diameter than mice. In conclusion, mice and rats present distinct tissue mechanical properties, which are accompanied by specific extracellular fiber composition.     

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.     

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.     

Development of elastic-system fibers in human vocal cords

The morphogenesis of elastic fibers in human, fetal and adult vocal cords was studied by light microscopy and transmission electron microscopy. The elastic system includes elastic, elaunin and oxytalan fibers at different stages. The development of elastic-system fibers in human vocal cord is characterized by every stage of maturation, whether normal, stifled or accelerated, according to areas.     

Phosphatidylinositol-dependent bond between alkaline phosphatase and collagen fibers in the periodontal ligament of rat molars

Alkaline phosphatase (ALP) is anchored to the outer leaflet of the lipid bilayer via phosphatidylinositol (PI) and ALP activity has been localized in the plasma membrane of numerous tissues. In the periodontal ligament ALP activity is found in the collagen fibers in addition to the plasma membrane of the osteoblasts and fibroblasts. In this study, we examined the distribution of ALP activity in the periodontal ligament of rat molars and also examined whether the bond between ALP and collagen fibers is dependent on PI by using phosphatidylinositol-specific phospholipase C (PI-PLC). ALP activity was distributed in the periodontal ligament. The activity mirrored the distribution of collagen fibers in the periodontal ligament. Cytochemical analysis also demonstrated that ALP activity was located not only in the plasma membrane of fibroblasts, but also in the collagen fiber bundles and fibrils in the periodontal ligament. After treatment with PI-PLC, the loss of ALP activity in the periodontal ligament was observed histochemically, and the loss of ALP activity in the fibroblasts as well as in the collagen fiber bundles and fibrils was observed cytochemically. These results strongly indicate that the bond between ALP and the collagen fibers is also dependent on PI.     

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.     

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.     

Lung tissue mechanics and extracellular matrix composition in a murine model of silicosis.

The dynamic mechanical properties of lung tissue and its contents of collagen and elastic fibers were studied in strips prepared from mice instilled intratracheally with saline (C) or silica [15 (S15) and 30 days (S30) after instillation]. Resistance, elastance, and hysteresivity were studied during oscillations at different frequencies on S15 and S30. Elastance increased from C to silica groups but was similar between S15 and S30. Resistance was augmented from C to S15 and S30 and was greater in S30 than in S15 at higher frequencies. Hysteresivity was higher in S30 than in C and S15. Silica groups presented a greater amount of collagen than did C. Elastic fiber content increased progressively along time. This increment was related to the higher amount of oxytalan fibers at 15 and 30 days, whereas elaunin and fully developed elastic fibers were augmented only at 30 days. Silicosis led not only to pulmonary fibrosis but also to fibroelastosis, thus assigning a major role to the elastic system in the silicotic lung.     

Distribution of elastic system fibers in the peripheral nerves of mammals

Various nerves of 6 representative species of mammals (including the human) were studied by the comparative association of the selective staining methods of light microscopy with the ultrastructural observation after tannic acid-glutaraldehyde fixation, which provided a reliable means of characterizing the different elastic system fibers. Although mature elastic fibers are not present in nerves, elastic-related fibers are frequently observed: oxytalan fibers are found mainly in the endoneurium, whereas elaunin fibers predominate in the epineurium. These fibers are longitudinally disposed, in a parallel orientation to the axons. The fact that these findings were consistently observed in the nerves of all species studied argues strongly in favor of the existence of a uniform structural pattern of distribution of elastic system fibers in nerves as a general phenomenon in mammals.     

Elastic tissue in the limiting membrane of the human seminiferous tubule

The structure and distribution of elastic tissue were studied in the limiting membrane of the seminiferous tubule from normal human testis. The elastic and elastic-related fibers (oxytalan and elaunin) were recognized by their tinctorial and ultrastructural characteristics. The connective structures of the limiting membrane, including the fibrotubules and the amorphous material of the elastic system, were studied after tannic acid-glutaraldehyde fixation. Fibrotubules (oxytalan fibers) were observed in almost all intercellular spaces of the limiting membrane, closely related to the contractile cells; the elaunin fibers (patches of amorphous material surrounded by bundles of fibrotubules) were evident in the outermost layers. The function of this system of elastic tissue and myoid cells is discussed, considering the permeability membrane and the role of the myoid cells in the elastogenesis and contractility of the seminiferous tubule.     

Ultrastructure of the venous ampulla in the interradicular microvascular bed of the mandibular molars of Mus musculus

The interradicular periodontal ligament of mandibular molars contains an apparently unique dilated vessel straddling the interradicular alveolar bone. This structure is designated a venous ampulla. The vessel possesses a luminal length and width of approximately 200 X 100 microns, respectively. Ultrastructurally, the endothelium has an average thickness of 0.35 micron, a continuous basement membrane, and an incomplete layer of pericytes. Open endothelial junctions are not present. The anatomy of the vessel wall differs markedly on the dental- and bone-related aspects. Calculated ratios for the luminal diameter to wall thickness vary from 1:80 to 1:150. Postcapillary-sized limbs from this vessel drain into the interradicular septum of bone and the ligament microvascular bed. Arterial supply to the ampulla is provided via arteriovenous anastomoses characterized by their association with myelinated and unmyelinated nerve groups. Oxytalan fibers are present throughout the wall of the venous ampulla, penetrating to the abluminal side of the endothelium where they are associated with unmyelinated axons and free nerve endings. Elsewhere, oxytalan fibers are related to the arteriovenous anastomoses and their accompanying myelinated and unmyelinated nerves located adjacent to the endothelium. Pericytes form membranous contacts with the endothelium of the arteriovenous anastomoses and have processes penetrating the endothelium basement membrane.