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.     

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.     

STRUCTURAL BIOLOGY OF THE FIBRES OF THE COLLAGENOUS AND ELASTIC SYSTEMS

The different types of fibres of the collagenous and elastic systems can be demonstrated specifically in tissue sections by comparing the typical ultrastructural picture of each of the fibre types with studies using selective staining techniques for light microscopy. A practicalmodus operandi, which includes the recommended staining procedures and interpretation of the results, is presented. Micrographs and tables are provided to summarize the differential procedures. Reticulin fibres display a distinct argyrophilia when studied by means of silver impregnation techniques, and show up as a thin meshwork of weakly birefringent, greenish fibres when examined with the aid of the Picrosirius-polarization method. In addition, electron-microscopic studies showed that reticulin fibres are composed of a small number of thin collagen fibrils, contrasting with the very many thicker fibrils that could be localized ultrastructurally to the sites where non-argyrophilic, coarse collagen fibres had been characterized by the histochemical methods used. The three different fibre types of the elastic system belong to a continuous series: oxytalan—elaunin—elastic (all of the fibre types comprising collections of microfibrils with, in the given sequence, increasing amounts of elastin). The three distinct types of elastic system fibres have different staining characteristics and ultrastructural patterns. Ultrastructurally, a characteristic elastic fibre consists of two morphologically different components: a centrally located solid cylinder of amorphous and homogeneous elastin surrounded by tubular microfibrils. An oxytalan fibre is composed of a bundle of microfibrils, identical to the elastic fibre microfibrils, without amorphous material. In elaunin fibres, dispersed amorphous material (elastin) is intermingled among the microfibrils.     

Histochemistry of elastic and related fibres in the human eye in health and disease

Summary Although the presence of mature elastic fibres in the sclera, trabecular meshwork and Bruch's membrane of the human eye has been recognized for many years, it is only latterly that the existence of the elastic-related fibres oxytalan and elaunin has been appreciated. The microfibrillar component of elastic, oxytalan, which is present in several ocular structures in infancy, can either mature to fully-developed elastic tissue or, as in the cornea, disappear in subsequent years. Deposition of elastic fibres, particularly the incomplete forms, can occur in the post-developmental period in a variety of disease states but the stimulus and functional significance in most situations is obscure. The evidence suggests, however, that the capacity to form elastic-related tissue is not confined to any one cell type.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

Preservation of elastic system fibers and of collagen molecular arrangement and stainability in an Egyptian mummy

The present findings show that both elastic system fibers and collagen markedly resisted change in tissues more than 2000 years old. The distribution of elastic fibers and elastic-related fibers (namely, oxytalan and elaunin fibers) in mummified tissues coincided with the observations made on the modern human tissues used as controls. The collagenous structures present in tissue sections obtained from the Egyptian mummy studied took on a deeply red colour when stained in the Picrosirius solution indicating that, as well as in the fresh controls, the basic groups in the collagen molecules were available for reacting with the strongly acidic dye Sirius Red. When viewed with polarized light, the collagen in the same tissue sections displayed an increased birefringence, which shows that the collagen molecules in mummified tissues maintain the oriented disposition which is typical of the modern human tissues used as controls. The methods employed have proved to be useful for the delineation of the elastic system fibers and of the collagenous scaffolding, which may be used as valuable landmarks in the study of the histoarchitecture of organs that have undergone considerable distortion.     

Preservation of elastic system fibers and of collagen molecular arrangement and stainability in an Egyptian mummy

Summary The present findings show that both elastic system fibers and collagen markedly resisted change in tissues more than 2000 years old.The distribution of elastic fibers and elastic-related fibers (namely, oxytalan and elaunin fibers) in mummified tissues coincided with the observations made on the modern human tissues used as controls.The collagenous structures present in tissue sections obtained from the Egyptian mummy studied took on a deeply red colour when stained in the Picrosirius solution indicating that, as well as in the fresh controls, the basic groups in the collagen molecules were available for reacting with the strongly acidic dye Sirius Red. When viewed with polarized light, the collagen in the same tissue sections displayed an increased birefringence, which shows that the collagen molecules in mummified tissues maintain the oriented disposition which is typical of the modern human tissues used as controls.The methods employed have proved to be useful for the delineation of the elastic system fibers and of the collagenous scaffolding, which may be used as valuable landmarks in the study of the histoarchitecture of organs that have undergone considerable distortion.Supported in part by Grant no. 43.83.0610/00 from Financiadora de Estudos e Projetos (FINEP-FNDCT). G.S. Montes is Career Investigator of the Brazilian National Research Council (CNPq)     

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.     

Tendon tissue microdamage and the limits of intrinsic repair

The transmission of mechanical muscle force to bone for musculoskeletal stability and movement is one of the most important functions of tendon. The load-bearing tendon core is composed of highly aligned collagen-rich fascicles interspersed with stromal cells (tenocytes). Despite being built to bear very high mechanical stresses, supra-physiological/repetitive mechanical overloading leads to tendon microdamage in fascicles, and potentially to tendon disease and rupture. To date, it is unclear to what extent intrinsic healing mechanisms of the tendon core compartment can repair microdamage. In the present study, we investigated the healing capacity of the tendon core compartment in an ex vivo tissue explant model. To do so, we isolated rat tail tendon fascicles, damaged them by applying a single stretch to various degrees of sub-rupture damage and longitudinally assessed downstream functional and structural changes over a period of several days. Functional damage was assessed by changes in the elastic modulus of the material stress-strain curves, and biological viability of the resident tenocytes. Structural damage was quantified using a fluorescent collagen hybridizing peptide (CHP) to label mechanically disrupted collagen structures. While we observed functional mechanical damage for strains above 2% of the initial fascicle length, structural collagen damage was only detectable for 6% strain and beyond. Minimally loaded/damaged fascicles (2–4% strain) progressively lost elastic modulus over the course of tissue culture, despite their collagen structures remaining intact with high degree of maintained cell viability. In contrast, more severely overloaded fascicles (6–8% strain) with damage at the molecular/collagen level showed no further loss of the elastic modulus but markedly decreased cell viability. Surprisingly, in these heavily damaged fascicles the elastic modulus partially recovered, an effect also seen in further experiments on devitalized fascicles, implying the possibility of a non-cellular but matrix-driven mechanism of molecular repair. Overall, our findings indicate that the tendon core has very little capacity for self-repair of microdamage. We conclude that stromal tenocytes likely do not play a major role in anabolic repair of tendon matrix microdamage, but rather mediate catabolic matrix breakdown and communication with extrinsic cells that are able to effect tissue repair.     

Tendon crimps and peritendinous tissues responding to tensional forces

Tendons transmit forces generated from muscle to bone making joint movements possible. Tendon collagen has a complex supramolecular structure forming many hierarchical levels of association; its main functional unit is the collagen fibril forming fibers and fascicles. Since tendons are enclosed by loose connective sheaths in continuity with muscle sheaths, it is likely that tendon sheaths could play a role in absorbing/transmitting the forces created by muscle contraction. In this study rat Achilles tendons were passively stretched in vivo to be observed at polarized light microscope (PLM), scanning electron microscope (SEM) and transmission electron microscope (TEM). At PLM tendon collagen fibers in relaxed rat Achilles tendons ran straight and parallel, showing a periodic crimp pattern. Similarly tendon sheaths showed apparent crimps. At higher magnification SEM and TEM revealed that in each tendon crimp large and heterogeneous collagen fibrils running straight and parallel suddenly changed their direction undergoing localized and variable modifications. These fibril modifications were named fibrillar crimps. Tendon sheaths displayed small and uniform fibrils running parallel with a wavy course without any ultrastructural aspects of crimp. Since in passively stretched Achilles tendons fibrillar crimps were still observed, it is likely that during the tendon stretching, and presumably during the tendon elongation in muscle contraction, the fibrillar crimp may be the real structural component of the tendon crimp acting as shock absorber. The peritendinous sheath can be stretched as tendon, but is not actively involved in the mechanism of shock absorber as the fibrillar crimp. The different functional behaviour of tendons and sheaths may be due to the different structural and molecular arrangement of their fibrils.     

Evidence for basement membranes in rat tail tendon sheaths

The presence of anti-laminin antibodies and a basement membrane-like thin electrondense lamina has been demonstrated in the peritendineum of the rat tail tendon by indirect immunofluorescence and electron microscopy.