Areas of asbestoid (amianthoid) fibers in human thyroid cartilage characterized by immunolocalization of collagen types I,II, IX,XI and X

The distribution of type I, II, IX, XI and X collagens in and close to areas of asbestoid (amianthoid) fibers in thyroid cartilages of various ages was investigated in this study. Asbestoid fibers were first detected in thyroid cartilage from a 3-year-old male child. Areas of asbestoid fibers functionally appear to serve as guide rails for vascularization of thyroid cartilage. Alcian blue staining in the presence of 0.3 M MgCl₂ revealed a loss of glycosaminoglycans in areas of asbestoid fibers. In addition, the fibers reacted positively with antibodies against collagen types II, IX and XI, but showed no staining with antibodies to collagen types I and X. Territorial matrix of adjacent chondrocytes showed the same staining pattern. In addition to staining for type II, IX and XI collagens, asbestoid fibers showed strong immunostaining for type I collagen after puberty but not for type X collagen. However, groups of chondrocytes within areas of asbestoid fibers reacted strongly with antibodies to type X collagen, suggesting that this collagen plays an important role in matrix of highly differentiated chondrocytes. The finding that these type X collagen-positive chondrocytes also revealed immunostaining for type I collagen confirms previous studies showing that hypertrophic chondrocytes can further differentiate into cells that are characterized by the synthesis of type X and I collagens.     

Amianthoid (asbestoid) transformation: electron microscopical studies on aging human costal cartilage

The present study reports on the fine structure of human costal cartilage at different ages in order to obtain information on the morphogenesis of amianthoid fibers. Our results reveal an overall increase of collagen fibril diameter with increasing age, even in areas with no signs of amianthoid transformation. Ultrastructural evidence is presented that this increase in diameter is due to a gathering of the preexisting collagen fibrils. The age-related change in collagen fibril diameter is paralleled by changes in the composition and ultrastructural appearance of cartilage proteoglycans (as revealed by acridine orange staining). Acridine-orange-positive filaments indicative for proteoglycans are markedly reduced in size with advancing age in centrally located regions of costal cartilage. Treatment with testicular hyaluronidase previous to acridine-orange staining leaves these small proteoglycan filaments unaffected. By contrast, the filaments visible after acridine-orange staining in the extracellular matrix near to the perichondrium are susceptible to hyaluronidase treatment. Infrequently, a sharp increase in collagen fibril diameter can be observed in territorial matrix areas of degenerating chondrocytes. This observation is conspicuous at ages of 10 and 20 years. Amianthoid transformation is characterized by the appearance of collagen fibrils strictly arranged in parallel. These amianthoid fibers are embedded in a matrix rich in small acridine-orange-positive filaments similar to the proteoglycan filaments observed in centrally located matrix regions. It can be concluded that extensive remodelling not only of the collagen fibrils but also of the cartilage proteoglycans is involved in the development of amianthoid transformation.     

Collagen fibril diameters in arteries of mice. A comparison of manual and computer-aided morphometric analyses

Arteries of mice were studied by a silver impregnation technique, by the Picrosirius-polarization method and by transmission electron microscopy. The histochemical results obtained coincided with the electron-microscopic observations in showing the presence of two distinct collagen populations, segregated into different compartments of each artery. The fibrous component of the tunica media was comprised of reticulin fibers, which displayed a distinct argyrophilia when studied by means of the silver impregnation technique, and showed up as thin, weakly birefringent, greenish fibers when examined with the aid of the Picrosirius-polarization method. In addition, the electron-microscopic studies disclosed the presence of thin collagen fibrils in the tunica media, contrasting with the thicker fibrils that could be localized ultrastructurally to the tunica adventitia where nonargyrophil, coarse collagen fibers had been characterized by the histochemical methods used. In this respect, collagen distribution in arteries of mice is very similar to the pattern that was consistently observed in the other species studied, which argues in favor of the existence of a uniform structural pattern of collagen distribution that is a general phenomenon in vertebrate arteries. Experimental results comparing the traditional method and the computer-aided measurement of collagen fibril diameters showed that the system provides results equivalent to those produced by manual execution. In addition, the advantage in speed of the computer-aided method should prove useful in complicated studies where numerous structures are involved.     

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.     

The influence of tissue section thickness on the study of collagen by the Picrosirius-polarization method

Summary The influence of tissue section thickness on the color and intensity of birefringence displayed by collagen in tissue sections studied by means of the Picrosirius-polarization method, is reported in this paper. When dermal collagen sections of different thicknesses (ranging from 0.25 to 11 m) were studied by this method, it became evident that not only did the intensity of birefringence increase proportionally to tissue section thickness, as was to be expected, but also a gradual shift in color from green through yellow to red could be observed as tissue section thickness increased. The limitations of the Picrosirius-polarization method for the localization of collagen types I, II, and III in routinely used histological slides is discussed, showing that this method is useful for the study of the distribution of the different types of interstitial collagen in normal adult vertebrate organs.     

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)     

Ultrastructural analysis of collagen fibers in the conductive and working myocardium of the sinoatrial region of the human heart

Qualitative and quantitative electron microscopic analysis of collagenous fibers has been performed in the sinusal node (SN) and perinodal working myocardium of the right atrium (RA) in the hearts of 22 men, died at the age of 23-63 years. Five of them died from alcoholic cardiomyopathy, 8--from coronary cardiac disease and 9--from noncardiac causes and craniocerebral trauma (group of comparison). In 21 cases seven types of ultrastructural changes of the collagenous fibers have been revealed in the SN (changes in the form, size and degree of their osmiophilia). As a whole the volumetric density of the collagenous fibers in the selection makes 24.4 +/- 1.6% from the volume of the SN conductive myocardium and 5.7 +/- 0.8% from the volume of the RA perinodal working myocardium. Content of the collagenous fibers in the SN does not depend on the death cause, age of the dead or on degree of the cardiac hypertrophy. Content of the collagenous fibers in the RA is 2.2 times higher at a sudden coronary death, than in the group of comparison. With age in the RA the volumetric density of the collagenous fibers increases by 1.9 times, and with rise in the cardiac mass, it increases by 2.2 times. The data obtained are discussed in order to understand the role of the collagenous fibers in functioning of the sinoauricular area both normal and at the cardiac pathology.     

Metastasis to the breast from an adenocarcinoma of the lung with extensive micropapillary component: a case report and review of the literature

Intranodal palisaded myofibroblastoma (IPM) is a benign mesenchymal neoplasm originating from smooth muscle cells and myofibroblasts. It is characterized by spindle cells, amianthoid fibers, and by the proliferation of hemosiderin-containing histiocytes in the lymph node. A nodular lesion was excised from the inguinal region of an 80-year-old male patient. Macroscopic examination of a section of the lesion demonstrated a solid appearance with hemorrhagic areas. Microscopic examination revealed spindle cell proliferation, amianthoid fibers, hemosiderin pigment, and extravasated erythrocytes. Nuclei of the spindle cells displayed a palisaded appearance. Compressed lymphoid tissue was observed around the lesion. With Masson's trichrome, spindle cells stained as smooth muscle, whereas collagen staining was observed in homogeneous eosinophilic accumulations. Neoplastic cells were identified by the presence of vimentin and SMA. The Ki67 index was less than 1%. In light of these results, the case was diagnosed as "intranodal palisaded myofibroblastoma." IPM is an uncommon neoplasm originating from the stromal component of the lymph node. Although IPM is benign, it is frequently confused with metastatic lesions.     

A fine structural localization of the non-specific cholinesterase activity in glomerular nerve formations (endings)

Snout glabrous skin (rhinarium) of the cat is innervated not only by typical simple lamellar corpuscles but also glomerular formations. In contrast to simple lamellar corpuscles, glomerular nerve formations are located away the dermal papillae. In cross sections, glomerular nerve formation consists of several axonal profiles enveloped by 1-2 cytoplasmic lamellae of Schwann cells. The space among them is filled by collagenous microfibrils and the basal lamina-like material. Capsule was composed from fibroblast-like cells without definite basal lamina. An electron-dense reaction product due to non-specific cholinesterase activity was associated with Schwann cells and their processes surrounding unmyelinated terminal portion of the sensory axons. Abundant reaction product was bound to the collagenous microfibrils and was deposited in extracellular matrix between Schwann cell processes. These results are further evidence for the presence of the non-specific cholinesterase molecules as integral component of the extracellular matrix in sensory corpuscles. On the basis of histochemical study two possible explanation are considered for functional involving of this enzyme in sensory nerve formations.     

Evidence for collagen molecular orientation in basement membranes

Summary The following basement membranes (BMs) from representative species of the main vertebrate classes were studied by the Picrosirius-polarization method: lens capsule, Reichert's membrane and glomerular BMs. A distinct birefringence was consistently observed in all BMs from all species studied by this method. The results reported provide a strong evidence for collagen macromolecular orientation in BMs. Heparitin sulphate was the only glycosaminoglycan detected in dog lens capsules.     

Detection of the most common G6PD gene mutations in Chinese using amplification refractory mutation system.

Glucose-6-phosphate dehydrogenase (G6PD) is the most common human enzymopathy. To date more than 122 mutations in the G6PD gene have been discovered, among which 12 point mutations are found in the Chinese. The 2 most common mutations, G1388A and G1376T, account for more than 50% of mutations representing various regions and ethnic groups in China. Setting up a simple and accurate method for detecting these mutations is not only useful for studying the frequency of the G6PD genotypes, but also for finding new mutations. The purpose of this study was to find a simple, inexpensive and accurate method for detecting these common mutations. The amplification refractory mutation system (ARMS) method was used in this study. Samples from 28 G6PD-deficient males were investigated. The natural and mismatched amplification and restriction enzyme digestion method was used as a standard method to evaluate the nature of the point mutations. Sixteen cases were found carrying the G1388A mutation and 12 the G1376T mutation. Fourteen cases of G1388A and 10 cases of G1376T were confirmed by ARMS. Four cases were not in concordance with the results obtained by the mismatched amplification-restriction enzyme digestion. These 4 cases were then judged by direct PCR sequencing at exon 12. The DNA sequencing data supported the results obtained by ARMS. Thus we concluded that the ARMS is a rapid, simple, inexpensive and accurate method for detecting the most common G6PD gene mutations among the Chinese.     

The collagen of the vertebrate peripheral nervous system

Summary Nerves and ganglia from a variety of fish, amphibian, reptilian and mammalian species were studied by optical and electron microscopy. Observations using the Picrosirius-polarization method strongly suggest that two different types of collagen fibers are present in the connective tissues of nerves and ganglia. Electron microscopy of nerves and ganglia showed the presence of two different collagen fibril populations, distinguishable on the basis of diameter, located in different compartments of these structures. Thicker fibrils are present in nerve and ganglionic epineurium. Thinner fibrils are present in the endoneurium, surrounding nerve fibers and ganglionic cells, and between the concentric layers of perineurial cells. These results were consistently observed in all species studied and very probably represent a general phenomenon in vertebrates.This work was aided by a grant from the Fundação de Amparo à Pesquisa do Estado de São Paulo     

Structuro-functional organization of the fiber network in the human Achilles tendon

Owing to a complex morphological investigation of the human Achilles tendon, it was possible to distinguish four levels of the structural-functional organization of its fibrous elements and to reveal some regularities of their structure that recur at all the levels. Thus, collagenous molecules, microfibrillae, fibrillae and fibers have a wavy-spiral conformation. This spatial form is stabilized by a complex or a system of transversal connections corresponding to the given level of the organization. In order to maintain integrity (the structural-functional unity) of each level, certain substances of polysaccharide nature take part. Along the course of the long tendinous axis, a re-distribution (branching) of the fibrillar elements is observed at all the levels of the structural-functional organization.     

Elastoid Changes in the Gingiva of Aged Humans

Elastotic changes were demonstrable in the gingivae of both dentulous and edentulous jaws obtained from both male and female humans varying in age from 62–92 years. Sections of gingivae from all the aged individuals exhibited numerable thick fibers that stained positive with iron hematoxylin or orcein. These positive staining fibers were found in the lamina propria radiating into the connective tissue papillae, coursing throughout the zona reticularis, as well as appearing as black amorphous masses. Pretreatment of sections with acid hydrolysis before staining by the two elastic tissue stains led to a loss of stainable fibers for elastin. In contrast the gingivae of a young adult did not contain elastic positive fibers as seen in the aged gingivae. The thick elastotic fibers found in the aged gingivae were argyrophilic in nature when the sections were impregnated with silver nitrate indicating that they were collagenous in nature. It is felt that the elastoid-like fibers in aging gingiva are another phase in the altering of collagen during the aging process.     

Functional architecture of the splenic vein in the adult human

The spatial arrangement of the muscular, elastic and collagenous fibers was examined in 20 splenic veins of adult humans. Histologic and panorganographic sections were stained by the following methods: azan, resorcinfuchsin (Weigert), Verhoeff-van Gieson's stain and Masson's trichrome. Unstained sections were examined under polarized light. The results show that the muscular and collagenous fibers of the splenic vein exhibit an elongated spiral arrangement with no polarization. The elastic fibers form a threedimensional network disposed predominantly in a longitudinal direction. Hemodynamics are discussed based on the spatial structure of the muscular, elastic and collagenous elements of the vein wall and its relationship with the circulatory functions of the spleen.     

A structural model used to evaluate the changing microstructure of maturing rat skin

A microstructurally based model for soft biological tissues was adapted to model the non-linear response of the collagenous network in skin. Quasi-static uniaxial tensile tests were conducted on dorsal skin of rats aged 1-4 months. Specimens were taken longitudinally and transversely (laterally) to the spine and tested at 1.5% s-1. The model showed fiber stiffness to increase laterally during maturation and to be greater laterally than longitudinally. It also indicated the migration of the 'heel' portion of the response curve toward the origin during maturation to be due to less crimping in the fibers. These results were qualitatively supported by microscopical observations. The model was found to be well suited to fit response curves exhibiting a well-defined 'heel' region. For curves exhibiting a more flat, featureless 'heel' region (typical of 1 month longitudinal specimens) the model was not well suited. The results of this study indicate that this simple three-parameter model might be a useful tool in future studies to analyze pathological changes in the collagenous microstructure.     

Elastic fibers in the anulus fibrosus of the dog intervertebral disc

A light microscopic investigation of the anulus fibrosus in cervical intervertebral discs of the dog was conducted to ascertain the arrangement and distribution of elastic fibers. Elastic fibers were observed in all lamellae of the anulus fibrosus. However, collagenous fibers were the predominant type of connective tissue fiber, and elastic fibers were randomly dispersed among them. Intralamellar (collagenous and elastic) fibers were vertically and obliquely oriented in both superficial and deep lamellae of the anulus fibrosus. All intralamellar fibers were densely and regularly arranged in superficial lamellae, but they were more loosely organized in deep lamellae. A narrow border of interlamellar, elastic fibers was observed between broader, contiguous lamellae in the superficial zone of the anulus fibrosus. Interlamellar elastic fibers wer vertically and obliquely arranged in superficial lamellae; however, they were radially oriented in deep lamellae. The deepest lamella of the anulus fibrosus consisted of a loose, three-dimensional network of intermeshing collagenous and elastic fibers. These observations suggest that elastic fibers are integral components of the articular and shock absorption mechanisms of the anulus fibrosus, and the cervical intervertebral disc of the dog is a suitable model for experimental investigation of the role of elastic fibers in intervertebral disc herniation.     

Ultrastructure of the pulmonary trunk wall during the prenatal period of ontogenesis

By means of transmissive electron microscopy stages of morphogenesis have been studied in the pulmonary trunk wall of 12 healthy human embryos and fetuses of 5-24-week-old. There is a close dependence in development of the smooth muscle component of the middle tunic, elastic and collagenous fibers of the pulmonary trunk wall. In the early prenatal morphogenesis of the pulmonary trunk fibrillogenesis develops intensively, overtakes the differentiation time of myocytes from mesenchymal cells, the collagenous fibers appearing earlier than the elastic ones. The structures, that ensure solidity of the vascular wall (collagenous fibers), appear in embryogenesis earlier that those, ensuring its elasticity (ability to reversible deformity).     

Quantitative Characterization of Collagen in the Fibrotic Capsule Surrounding Implanted Polymeric Microparticles through Second Harmonic Generation Imaging

The collagenous capsule formed around an implant will ultimately determine the nature of its in vivo fate. To provide a better understanding of how surface modifications can alter the collagen orientation and composition in the fibrotic capsule, we used second harmonic generation (SHG) microscopy to evaluate collagen organization and structure generated in mice subcutaneously injected with chemically functionalized polystyrene particles. SHG is sensitive to the orientation of a molecule, making it a powerful tool for measuring the alignment of collagen fibers. Additionally, SHG arises from the second order susceptibility of the interrogated molecule in response to the electric field. Variation in these tensor components distinguishes different molecular sources of SHG, providing collagen type specificity. Here, we demonstrated the ability of SHG to differentiate collagen type I and type III quantitatively and used this method to examine fibrous capsules of implanted polystyrene particles. Data presented in this work shows a wide range of collagen fiber orientations and collagen compositions in response to surface functionalized polystyrene particles. Dimethylamino functionalized particles were able to form a thin collagenous matrix resembling healthy skin. These findings have the potential to improve the fundamental understanding of how material properties influence collagen organization and composition quantitatively.     

Diffusional anisotropy in collagenous tissues: fluorescence imaging of continuous point photobleaching

Molecular transport in avascular collagenous tissues such as articular cartilage occurs primarily via diffusion. The presence of ordered structures in the extracellular matrix may influence the local transport of macromolecules, leading to anisotropic diffusion depending on the relative size of the molecule and that of extracellular matrix structures. Here we present what we believe is a novel photobleaching technique for measuring the anisotropic diffusivity of macromolecules in collagenous tissues. We hypothesized that macromolecular diffusion is anisotropic in collagenous tissues, depending on molecular size and the local organization of the collagen structure. A theoretical model and experimental protocol for fluorescence imaging of continuous point photobleaching was developed to measure diffusional anisotropy. Significant anisotropy was observed in highly ordered collagenous tissues such as ligament, with diffusivity ratios >2 along the fiber direction compared to the perpendicular direction. In less-ordered tissues such as articular cartilage, diffusional anisotropy was dependent on site in the tissue and size of the diffusing molecule. Anisotropic diffusion was also dependent on the size of the diffusing molecule, with greatest anisotropy observed for larger molecules. These findings suggest that diffusional transport of macromolecules is anisotropic in collagenous tissues, with higher rates of diffusion along primary orientation of collagen fibers.