Lamina Fibroreticularis Research Articles

Ultrastructural and immunocytochemical characterization of ameloblast-enamel adhesion at maturation stage in amelogenesis in Macaca fuscata tooth germ.

Maturation-stage ameloblasts are firmly bound to the tooth enamel by a basal lamina-like structure. The mechanism underlying this adhesion, however, remains to be fully clarified. The goal of this study was to investigate the mechanism underlying adhesion between the basal lamina-like structure and the enamel in monkey tooth germ. High-resolution immunogold labeling was performed to localize amelotin and laminin 332 at the interface between ameloblasts and tooth enamel. Minute, electron-dense strands were observed on the enamel side of the lamina densa, extending into the degrading enamel matrix to produce a well-developed fibrous layer (lamina fibroreticularis). In un-demineralized tissue sections, mineral crystals smaller than those in the bulk of the enamel were observed adhering to these strands where they protruded into the surface enamel. Immunogold particles reactive for amelotin were preferentially localized on these strands in the fibrous layer. On the other hand, those for laminin 332 were localized solely in the lamina densa; none were observed in the fibrous layer. These results suggest that the fibrous layer of the basal lamina-like structure is partly composed of amelotin molecules, and that these molecules facilitate ameloblast-enamel adhesion by promoting mineralization of the fibrous layer during the maturation stage of amelogenesis.

A Special Construction of Subepidermal Capillary Loops in the Hippopotamus (Hippopotamus amphibius)

Based on LM, TEM, and histochemical methods, the study describes the specific structure of subepidemal capillary loops in the integument of the hippopotamus (Hippopotamus amphibius). At 25- 60 µm, the diameter of the capillaries was more than twenty times larger than those found in other mammals, as was the diameter of the epidermal contact area of the hairpin turn, which had enlarged up to 200-400 µm(2). At about 13,400, the number of loops per cm(2) was three times higher than in the few other mammalian species measured to date. The remarkable sheath (thickness 2- 20 µm) of the capillary loops consists of a multitude of fine collagen IV fibres, which were in direct contact with the epidermal stratum (str.) basale, emphasizing an origin from the lamina fibroreticularis of the basement membrane. Additionally, the sheath contained many regions filled with free fatty acids. All observations confirmed the view that the walls of the subepidermal capillaries in the hippopotamus are adapted to withstand high blood pressure, permitting a high rate of blood vesselbased heat transfer from the periphery of the body. Until now this function is only known as an important thermoregulatory response in highly active mammals, e.g. dolphins. However, under hot climatic conditions but without strong exercise for cooling, such ability could be an effective and energy-saving procedure in semi-aquatic mammals.

The human corneal epithelium after alcohol delamination: a structural and ultrastructural study

Dilute alcohol is one of the most popular methods for corneal epithelial removal during photorefractive keratectomy (PRK) and laser subepithelial keratomilieusis (LASEK). Even if the technique is used by nearly fifteen years, no concordant data are available on the effects of the exposition to dilute alcohol on the corneal epithelium. As in LASEK the epithelial flap obtained by the previous delamination is repositioned to improve corneal recovery, aim of the present work was to investigate the structure and the ultrastructure of the corneal epithelium after alcohol delamination. Ten patients undergoing PRK for myopic correction were consecutively included in the study. A 9-mm diameter cone was placed on the anaesthetized cornea and it was filled with 25% ethanol in BSS for 25 seconds. The cone was emptied and the corneal surface was washed off with BSS. The epithelial layer was lifted with beaver blade, peeled off with forceps, and processed for light (LM) and transmission electron microscopy (TEM). With LM, whilst superficial and wing cells showed a normal appearance, basal cells had significantly different staining patterns. In fact, in the same microscopic field they showed either normal morphology or paler nuclei and cytoplasm. When the specimens were observed with the TEM, all epithelial cells showed well-preserved intercellular spaces and junctional complexes. In the superficial cells perinuclear vacuolizations were present, whilst wing cells demonstrated no evident morphological changes. Clear basal cells had roundish nuclei with pale chromatin and clear cytoplasm with perinuclear endoplasmic reticulum and mitochondria and a large number of tonofilaments. Their basal membrane was generally intact, with many hemidesmosomes adhering to the basement membrane, which was formed only by the laminae lucida and densa. Dark basal cells showed irregular nuclei with condensed chromatin, vacuolated cytoplasm and few basal hemidesmosomes. Alcohol debridement can be considered as a valuable technique for removing corneal epithelium before PRK or for preparing an epithelial flap before LASEK: in fact it affects the binding of hemidesmosomes to the underlying basement membrane so that the lamina densa is separated from the lamina fibroreticularis. However, the observation of structural and ultrastructural changes of the basal cells, similar to those demonstrated in epithelial flaps obtained with the epikeratome, indicates the need for further studies to evaluate the corneal toxicity of the ethanol.

Histological evaluation of mechanical epithelial separation in epithelial laser in situ keratomileusis

To evaluate the effect of mechanical epithelial separation with an epikeratome on the histologic ultrastructure of epithelial flaps and stromal beds from human corneas. Departments of Ophthalmology, Osaka University Medical School, Osaka, and Tohoku University School of Medicine, Sendai, and Institute of Advanced Biomedical Engineering and Science and Medical Research Institute, Tokyo Women's Medical University, Tokyo, Japan. Eye-bank eyes were deepithelialized using an Epi-K epikeratome. Epithelial flaps and stromal beds were assessed by light and electron microscopy. Immunofluorescence staining for types IV and VII collagens, integrins alpha(6) and beta(4), and laminin 5 was also performed. Four eyes were evaluated. On scanning electron microscopy, the cleavage planes of epithelial flaps and stromal beds were relatively smooth. On transmission electron microscopy, epithelial flaps were separated partially within the lamina fibroreticularis and partially within the lamina lucida. Immunofluorescence showed positive staining for type VII collagen and discontinuous staining for type IV collagen in stromal beds. Discontinuous linear staining for types IV and VII collagens was observed in epithelial flaps. Staining for integrins alpha(6) and beta(4) was positive in some regions and discontinuous in other regions of epithelial flaps. In stromal beds, integrins alpha(6) and beta(4) had a patchy expression pattern. Staining for laminin 5 was intermittently positive along the basal side of epithelial flaps and stromal beds. Epithelial flaps created with an epikeratome were mechanically separated partly within the lamina fibroreticularis and partly within the lamina lucida. Stromal beds had relatively smooth surfaces with no obvious trauma to Bowman layer.

Basement membrane heterogeneity during chick development as shown by tomato (Lycopersicon esculentum) lectin binding.

Basement membranes (BMs) constitute a distinct compartment of the extracellular matrix (ECM). All BMs show a similar structural appearance but differ in molecular composition. These variations have critical functional implications. The aim of this study is to establish the pattern of the tomato lectin (Lycopersicon esculentum agglutinin--LEA) binding sites in the BMs of the developing chick embryo (stages 4-21, Hamburger and Hamilton, 1951) in order to achieve a better understanding of the molecular heterogeneity of BMs. The study was performed with transmission electron microscopy (TEM) histochemistry, and confocal laser microscopy. TEM showed that LEA bound to the lamina densa and to the lamina fibroreticularis of the BMs. Through the period studied, most of the LEA binding appeared in the ectodermal BM and its derivatives. In the limb bud, LEA binding to the ectoderm BM was more intense in the ventral half than in the dorsal half. Furthermore, LEA allowed the early (HH16) detection of the transverse fibrillar tracts. In the lens and in the inner ear primordium, the BMs were LEA positive through the placode and cup stages. The binding was progressively reduced through the vesicle stage. The BMs of the olfactory primordium, and of the Rathke's pouch were positive. In contrast, the BMs of the developing central nervous system were negative. The BMs of both the paraxial and the lateral plates of the mesoderm were negative, whereas the notochord and the BM of the Wolffian duct were positive. The endodermal BM and its derivatives were negative. The ECM located between the fusing endocardial tubes, and the BM of the fusion zone of the paired aortae, were positive. This suggested an active role of the LEA-positive glycoproteins in the fusion of endothelia. Our results show the heterogeneity of the chick embryo BMs during development. In addition, LEA constitutes an excellent marker for the primordial germ cells.

Ultrastructure of basement membranes in monkey and shark teeth at an early stage of development

The basement membrane, which separates the inner enamel epithelium from the dental papilla in the early stages of tooth development, is known to play a significant role in odontogenesis. In this review article, this basement membrane was described in detail based on our recent findings with the use of high-resolution electron microscopy. Tooth germs of a monkey (Macaca fuscata) and a shark (Cephaloscyllium umbratile) were processed for thin-section observations. During the early stage of development, the basement membrane of the inner enamel (dental) epithelium was composed of a lamina lucida, lamina densa, and much wider lamina fibroreticularis. At higher magnification, the lamina densa in both species was made up of a fine network of cords, which are generally the main constituents of the basement membranes. In the monkey tooth, the lamina fibroreticularis was rich in fibrils, which were now characterized as basotubules, 10-nm-wide microfibril-like structures. The space between the basotubules was filled with a cord network that extended from the lamina densa. Dental papilla cell processes were inserted into the lamina fibroreticularis, and their surface was closely associated with numerous parallel basotubules via 1.5- to 3-nm-wide filaments. In the shark tooth during its early stage of development, the basotubules were absent in the lamina fibroreticularis and only narrow extensions, 60-90 nm wide and 1-2 microm long, of the cord network of the lamina densa were present. The dental papilla cells were immobilized by means of the binding of their processes to the extensions. These results indicate that basement membranes in both monkey and shark teeth at early stage of development are specialized for functions as anchoring and firm binding, which are essential for the successful differentiation of the odontoblasts.

Scanning electron microscopic observations of the basement membranes with dithiothreitol separation.

Scanning electron microscopic (SEM) studies of the interstitial surface of the lamina densa can be performed with dithiothreitol separation, which is the only method of exposing this surface. SEM observation revealed the three-dimensional structures of the meshwork in the lamina densa and anchoring fibrils in dithiothreitol-separated specimens. Detection of the components of the basement membrane can be performed by immunoscanning electron microscopy on this exposed surface by comparing the backscattered and the secondary electron images. SEM observation also revealed the fine structure of the lamina fibroreticularis using separated dermis or the lamina propria mucosae.

An ultrastructural study of connective tissue in mollusc integument: II. Gastropoda

We studied the ultrastructure of the subepidermal connective tissue (SEC) in different zones of the integument in terrestrial, marine and freshwater gastropods (eight species). In all cases, the SEC was a layer of loose connective tissue between the basal membrane (BM) of the epidermis and the connective tissue of the deeper muscle layers. It was of monotonous structure and not differentiated into layers such as are found in mammalian dermis. The extracellular matrix (ECM) consisted of a network of collagen fibrils of variable diameter, with abundant anchoring devices and proteoglycans. In six species, variables quantities of haemocyanin were present within haemocoelic sinuses present in the SEC. The thickness and density of the BM varied from species to species, as well as within species in the various zones of integument. The ultrastructure of the lamina densa (LD) was indistinguishable from that of BM in bivalves and similar to that in mammals, although basotubules and double pegs were absent. An irregularly spaced lamina lucida was usuallypresent and was often shot thorough with filaments and small protrusions of the LD that connected with epithelial plasma membrane or with hemidesmosomes. A lamina fibroreticularis wasnot present. LD protrusions characterize the connection between BM and the ECM of SEC. In theterrestrial gastropods, a spongy matrix with ultrastructure closely similar to LD occupied large tracts of the SEC. In the mantle region of Arion rufus, the integumental SEC contained large cavities filled with spherical concretions, probably representing rudiments of a shell. In the mantle where theintegument contained abundant muscle fibres, the BM was thick and directly connected to the ECMof the SEC which consisted of compact laminae of collagen fibrils with abundant anchoring devices. Along the edge of the foot of Patella ulyssiponensis, the SEC contained a layer of paramyosinic muscle fibres adhering to the epidermis. No differences or gradations in integumental SEC structure could be related to the phylogenetic position of the species examined.

An ultrastructural study of connective tissue in mollusc integument: I. Bivalvia

The ultrastructure of the subepidermal connective tissue (SEC) in different areas of the integument of the bivalves Callista chione, Pecten jacobaeus, Mytilus galloprovincialis and Ostrea edulis was studied by transmission electron microscopy. The main organisation of the SEC was broadly similar in all species: the SEC was connected to the epidermis by a basement membrane and merged directly with the deeper connective tissue surrounding muscles. The SEC was not differentiated into layers like the papillary and reticular dermis of mammals, however, the architecture, thickness and shape of the basement membrane varied from species to species, as well as within species (in the foot, central or marginal zones of the mantle). The ultrastructure of the lamina densa was broadly similar to that in mammals: although basotubules and double pegs were absent, proteoglycans and rod-like units homologous to ‘double tracks’ were always abundant. A zone similar to the lamina lucida was irregularly present and was shot thorough with small protrusions of the lamina densa that connected with the epithelial hemidesmosomes or focal adhesions. Nevertheless zones were observed where the lamina densa fuse directly to the epithelial plasmamembrane. This variability of connection may be related to the various types of epidermal cell. A lamina fibroreticularis was not recognized since anchoring fibrils and microfibrils were not present; lamina densa protrusions into the extracellular matrix (ECM) of SEC characterize the connection between basement membrane and SEC. Collagen fibrils were small and of constant diameter and were never organised into fibres. Anchoring devices – similar to the anchoring plaques of mammalian dermis – were abundant and scattered between SEC collagen fibrils. The orange-pink pigmentation of C. chione seems due to electron-dense granules embedded within the connective ECM.

Ultrastructural verification of anchoring role of lamina fibroreticularis of dental basement membrane in odontogenesis.

In a previous study of the developing tooth a characteristic fibrillar layer associated with the basement membrane of the inner enamel epithelium was found to be a highly specialized lamina fibroreticularis of the basement membrane which is unusually rich in basotubules, 10 nm wide microfibril-like structures. In this study this layer was further examined in detail in the hope of ultrastructurally elucidating its role in odontogenesis. Tooth germs of the monkey (Macaca fuscata) were processed for thin section observations. Dental papilla cell processes were inserted into the lamina fibroreticularis and their surface was closely associated with numerous parallel basotubules. With high-resolution observations the space between the surface and nearest basotubules as well as the spaces between neighbouring basotubules were bridged by 1.5-3 nm wide filaments running perpendicular to the axis of basotubules. These results indicate that the processes of dental papilla cells are linked to groups of basotubules by means of 1.5-3 nm wide filaments. Immunoperoxidase staining showed the presence of fibronectin along basotubules as well as within the space between the process and basotubule. This result, together with the comparison of these filaments with microfibril-associated 1.2-3 nm wide fibronectin filaments and the reported abundance of fibronectin in the basement membrane area during odontogenesis, indicates that these 1.5-3 nm wide filaments are composed of fibronectin. After immunostaining for amyloid P component, done with the rat tissue because of the nature of an available antiserum, basotubules in the lamina fibroreticularis were positively stained, as has been shown in basotubules/microfibrils in other locations. Microfibrils function as anchoring rods by interlinking connective tissue components to one another and to the cells. Basotubules, thought to be basement membrane-incorporated microfibrils, in the lamina fibroreticularis in this study are also likely to function as an anchoring device to immobilize dental papilla cells along the basement membrane. Such an arrangement of mesenchymal cells is known to be crucial for the successful differentiation of odontoblasts in the developing tooth.

Alterations of Basal Lamina of the Seminiferous Epithelium during the Seasonal Suppression of Spermatogenesis in the Japanese Monkey (Macaca fuscata).

Primate spermatogenesis is characterized by yet unidentified mechanisms to regulate its rate-limiting process, spermatogonial proliferation. In the previous study using a seasonal breeder, Japanese monkey (Macaca fuscata), we demonstrated that the spermatogonial proliferation is suppressed through cytoplasmic liberation of PGP 9.5 when the spermatogenetic activity declines in the nonbreeding season. By comparing the morphology of the seminiferous tubules between the breeding and nonbreeding seasons, the present study demonstrates that in the nonbreeding season a basal lamina of seminiferous epithelium, with which spermatogonia directly keep contact, enlarges its extent at levels of lamina densa and lamina fibroreticularis, and is frequently invaginated. The invagination is composed of only the three laminae, not of cell components. The invaginations expand contact area between the spermatogonia and the basal lamina, which would interrupt spermatogonial proliferation. An ultrastructural distribution of laminin suggests that the invagination during the nonbreeding season is due to rearrangement of laminin molecules inside the basal lamina. The present results indicate that the alteration of the basal lamina of the seminiferous epithelium would mediate effects of the hormonal regulators and enable the fully restorable, transient suppression of spermatogenesis in primate.

Basement membrane in middle ear cholesteatoma. Immunohistochemical and ultrastructural observations.

We investigated the distribution of basement membrane zone (BMZ) components collagen type IV, collagen type VII, and fibronectin in human middle ear cholesteatoma, auditory meatal skin, and middle ear mucosa using both immunohistochemical and ultrastructural methods. Collagen type IV immunoreactivity of skin and middle ear mucosa is continuous in the BMZ, whereas cholesteatoma frequently showed absent immunoreactivity or focal discontinuities. Collagen type VII immunoreactivity is detected similarly within the BMZ of cholesteatoma and skin. Fibronectin immunoreactivity is observed within the dermoepithelial junction of skin and middle ear mucosa. In cholesteatoma, however, fibronectin immunoreactivity is markedly increased within the extrinsic BMZ and the subepithelial connective tissue. The ultrastructural arrangement of the BMZ of cholesteatoma is like that of skin; however, it exhibits distinct alterations of the lamina fibroreticularis and lamina densa. Our results outline cholesteatoma as a disease with disturbed cell matrix interactions analogous to those of wound reepithelialization.

Spatial distribution of enamel proteins and fibronectin at early stages of rat incisor tooth formation

Enamel proteins are secreted very early during amelogenesis, that is prior to mantle dentine formation, raising the possibility that they may participate in epithelial-mesenchymal interactions taking place during tooth development. These first enamel proteins associate with elements of the basement membrane interposed between the differentiating ameloblasts and odontoblasts. Fibronectin, a component of the basement membrane, is redistributed and accumulates along the apical portion of odontoblasts during their terminal differentiation. In order to determine whether any correlation exists between the redistribution of fibronectin and the secretion of the first enamel proteins, the spatial distribution of these two extracellular matrix proteins was examined during the presecretory stage of amelogenesis. Male Wistar rats were perfused with a formaldehyde-based fixative, and undemineralized and EDTA demineralized incisors were dehydrated in methanol and embedded in Lowicryl K4M resin. Ultrathin tissue sections were then processed for post-embedding, colloidal-gold immunocytochemistry with antibodies to enamel proteins, fibronectin or type III collagen. In the region of ameloblasts facing pulp, labelling for fibronectin was weak and mostly associated with the lamina fibroreticularis of the basement membrane separating differentiating ameloblasts and odontoblasts. As the mantle predentine formed the immunoreaction for fibronectin increased, particularly in the region of the basement membrane. Enamel proteins were also immunodetected in association with the lamina fibroreticularis and gradually accumulated as patches within mantle dentine and at its interface with ameloblasts. Von Korff collagen bundles, present between odontoblasts and in dentine, were immunolabelled for fibronectin and for type III collagen. Patches of granular material, immunoreactive for fibronectin and/or enamel proteins, were found along the odontoblastic processes and cell bodies. Although no evidence was obtained indicating a precise colocalization of fibronectin and enamel proteins, the results confirm that these two proteins can be found within similar extracellular compartments during mantle predentine-dentine formation. These data suggest that enamel proteins, by themselves or synergistically with other proteins, may play a part in the differentiation and/or formative events taking place at the ameloblast-odontoblast interface during the early stages of tooth development.

Light and electron microscopic localization of the alpha 1-chain and the E1 and E8 domains of laminin-1 in mouse kidney using monoclonal antibodies to establish the orientation of laminin-1 within basement membranes.

To localize the different domains of the laminin-1 molecule in tissues and gain insight into their in vivo relevance, we raised rat anti-mouse monoclonal antibodies (MAbs) against the entire molecule. Then we tested eight of the 20 clones producing anti-laminin-1 MAbs to specify their reactivity towards the alpha 1-, beta 1-, and gamma 1-chains and the elastase-cleaved fragments of the laminin-1 molecule. We found three MAbs with high titers in ELISA that showed good reactivity in embedded tissue. One of these reacted specifically against the E1 fragment, one against the E8 fragment, and one MAb detected the alpha 1-chain of laminin-1 but not the beta 1- or gamma 1-chain. All three MAbs are useful for light immunohistochemical investigations on cryosections and on paraffin-embedded material, and for ultrastructural localization of laminin-1 in LR Gold-embedded mouse tissue. Antibody staining of the E1 and E8 domains of laminin-1 revealed distinct localization of the molecule in the proximal tubule basement membranes of mouse kidney. The short arms (E1) of the laminin-1 molecule are predominantly located in the lamina lucida and the long arms (E8) are oriented towards the lamina fibroreticularis. Therefore, both MAbs are useful for studies of the orientation of the laminin-1 molecule in basement membranes. The distal tubule basement membranes did not show any distinct pattern of laminin-1 distribution. In general, the distal tubules showed the strongest reactions over the entire width of the basement membrane for all three MAbs. In contrast, the proximal tubule basement membranes showed somewhat weaker reactivity but a distinct pattern of laminin-1 distribution, with the E1 fragments oriented towards the adjacent epithelial cell surface.

Expression of basement membrane components in the dental papilla mesenchyme of monkey tooth germs--an immunohistochemical study.

The present work, which employs indirect immunoperoxidase methods, demonstrates electron microscopic localization of three major basement membrane (BM) components--type IV collagen, laminin, and heparan sulfate proteoglycan--at the early stages of odontogenesis in tooth germs of the Japanese macaque (Macaca fuscata). Intense immunostaining for each examined component occurred at the interface between the inner enamel epithelium and the dental papilla mesenchyme. At higher magnification, immunoreaction products were observed both in the lamina densa and lamina fibroreticularis. Fuzzy substances occurring very close to the lamina fibroreticularis manifested moderate immunoreactivity. In addition, immunostaining took place in the dental papilla mesenchyme. The dental papilla cells located close to the BM demonstrated immunoreactive material mainly on plasma membranes facing the BM. Reaction products were also observed in large concavities formed in some areas of the cell surfaces; and small, immunopositive vesicles occurred close to the plasma membrane. Immunoreaction products could be found in the cisternae of the rough endoplasmic reticulum of some mesenchymal cells. These findings suggest that dental papilla mesenchymal cells may produce the three major BM components and those of the components that are incorporated into the dental BM--particularly into the lamina fibroreticularis--during tooth development.

Die Basalmembran bei Erkrankungen der Stimmlippen: Elektronenmikroskopische und immunmorphologische Befunde*

The basement lamina of the epithelium of the true vocal cords of 34 inpatients suffering from chronic laryngitis, Reinke's oedema and squamous cell carcinoma have been investigated with the electron and immunofluorescence microscope. In chronic laryngitis, the lamina fibroreticularis is thickened (due to collagen type VII), corresponding to the clinical finding. In this layer mobile cells of the connective tissue can be found. In cases of Reinke's oedema it is the lamina densa which might be thickened. In this disease, also lamina densa-like material can protrude into the lamina fibroreticularis, and the number of anchoring filaments is increased. In squamous cell carcinoma we found the basement lamina irregularly arranged and folded. The lamina densa was always interrupted by numerous small gaps and in some areas the basement membrane could not be identified over a long distance. Lamina densa-like material was also found between the tumour cells within the epithelium. With the immunofluorescence microscope this material was proven as laminin, collagen type IV and heparan sulfate proteoglycan. Our investigation shows that malignant as well as benign lesions of the true vocal cords are characterised by distinct fine structural findings even concerning the basement lamina.

Characterization of the fibrillar layer at the epithelial-mesenchymal junction in tooth germs

A characteristic layer containing numerous fibrils is associated with the basement membrane of the inner enamel epithelium during the early stages of odontogenesis. However, its nature is not well understood. In this study, the layer was examined with high-resolution electron microscopy and immuno-histochemical staining. Tooth germs of monkeys (Macaca fuscata) were studied and each fibril in the layer was found to be a tubular structure, 8-9 nm in width, resembling a "basotubule", the tubular structure previously observed in various basement membranes. The space between the fibrils was filled with a network formed by irregular anastomosing strands with an average thickness of 4 nm; these strands resembled the "cords" forming the network in the lamina densa of basement membranes. After immunoperoxidase staining, fine threads immunoreactive for laminin staining were seen winding along the strands of the network, and 1.5-nm wide filaments, immunoreactive for type IV collagen, took the form of a network arrangement. The 5-nm-wide ribbon-like structures associated with the strands were identified as heparan sulfate proteoglycan by immunostaining. These results are similar to those obtained for the cord network of the lamina densa. The "fibrillar layer" therefore represents a highly specialized lamina fibroreticularis of the basement membrane of the inner enamel epithelium, and rich in basotubules.

Ultroser G and brain extract induce a continuous basement membrane with specific synaptic elements in aneurally cultured human skeletal muscle cells

Basement membrane (BM) components were studied on human muscle and skeletal muscle cells cultured on different media by immunofluorescence and electron microscopy. Their topographical relation with acetyl-choline receptors was investigated. Myotubes cultured on a combination of the serum substitute Ultroser G and brain extract show a continuous layer of heparan sulfate proteoglycans (HSPGs), laminin, and type IV collagen. In contrast, myotubes cultured on serum-containing media are associated with granular depositions of HSPG and laminin and only with wisps of type IV collagen. Omission of brain extract or substitution by chicken embryo extract results in an intermediate staining pattern. For all types of cultures, fibronectin is localized in and around mononuclear cells, but hardly associated with myotubes. A codistribution between clusters of acetylcholine receptors and HSPG and laminin and Vicia villosa B 4 lectin-positive material exists only in Ultroser G/brain extract-based myotubes like in muscle in vivo. No clustering is observed in serum-based myotubes. Electron microscopy reveals that the former myotubes are surrounded by a continuous BM consisting of a lamina lucida, lamina densa, and lamina fibroreticularis. Proteoglycans are present on the external site of the lamina densa and associated in a regular fashion with collagen fibrils. In conclusion, BMs associated with myotubes cultured on Ultroser G/brain extract resemble in many ways the in vivo situation, including synaptic specializations. Cultured myotubes may serve as a model system for studies on the structure and function of human muscular (synaptic) BM under normal and pathological conditions.

Invited Review: Structure and Composition of Basement Membranes and Other Basal Matrix Systems in Selected Invertebrates

Abstract Problems concerning terminology and relationships of basement membranes to extracellular matrices (ECMs) and connective tissue systems in invertebrates are discussed, and recommendations regarding nomenclature are presented. It is suggested to use ‘basal matrix’ as a comprehensive term for various basal ECM structures, and apply the term ‘basement membrane’ (BM) for a unique ECM structure that is fully resolved only by electron microscopy and as a general and descriptive word in both light microscopy, biochemistry and electron microscopy. When possible and relevant it is recommended to apply the subdivisions: ‘lamina lucida’, ‘lamina densa’ (= ‘basal lamina’) and ‘lamina fibroreticularis’ (sometimes missing). A brief and non‐technical account of the recent concepts of BM macromolecules and their assembly is given, based on mammalian models. Basement membranes probably first appeared in cnidarians, and from then on became obligatory ECM structures. It is emphasized that this event was crucial for evolution of the multicellular state, and for construction and morphogenesis of tissues and organs. Lower worms, such as turbellarians and nemertines, display the most varied and complex basal matrices. First, in annelids, complete correlation between BM structure and composition has been demonstrated, with isolation and consecutive localization of basement‐specific molecules such as, for example, collagen IV and laminin. Both in arthropods, especially insects, and echinoderms, the introduction of modern techniques corresponding to those used in mammalian BM research has now given equally valid results. An interesting and fundamental outcome of the reviewed work on invertebrate BMs is the realization of the early evolution of BM components. Macromolecules such as laminin, collagen IV and fibronectin are highly conserved, but with fine modulations, and in lower invertebrates they may exist without being assembled into true BM structures. Laminin and fibronectin are expressed with certainty in cnidarians, and fibronectin possibly already in sponges.

Ultrastructures of the epithelial basement membrane and the subepithelial capillaries in rabbit palatine tonsils.

The ultrastructure of both the epithelial basement membrane and the subepithelial capillaries in rabbit tonsils was investigated using light and transmission electron microscopy of sections, and scanning electron microscopy of alkali-water macerated tissues. The basement membrane of the crypt epithelium was seen to consist of the lamina lucida, lamina densa and lamina fibroreticularis. The lamina fibroreticularis is made up of both fine and thick collagen fibrils. The basement membrane possesses numerous pores (0.5-20 microns in diameter) through which free cells migrate. The basement membrane overlying the follicle protrudes hemispherically towards the crypt lumen, while that over the interfollicular area forms many papillary projections. The capillaries are surrounded by collagen fibrillar sheaths invariably located below the collagen fibrillar sheet of the epithelial basement membrane. The capillaries immediately below the crypt epithelium, including switch-back loops of capillaries in the papillae, are fenestrated sinusoids (20-40 microns in diameter). Plasma cells, lymphocytes and macrophages are numerously gathered around the capillaries. Possible functional relations between these free cells and the fenestrated sinusoids are discussed.