Hyaluronan in human deciduous tooth germs in the bell stage. Histochemistry and immunohistochemistry.

The primary aim of the present study was a localization of hyaluronan (HA) in human deciduous tooth germs in the bell stage. HA was compared to the content of chondroitin sulfates (CSs). HA was detected with a biotin-labeled HA-binding protein (HABP) and CS with a monoclonal antibody. As controls, enzyme digestions were carried out. Furthermore, the glycosaminoglycans were investigated histochemically with enzyme digestions followed by alcian blue staining. The investigation showed a considerable content of HA in the stellate reticulum, although CS was also found, primarily when treatment with protease was omitted. The dental papilla contained both HA and CS, while the predentin and the dentin contained only CS. The enamel did not contain any CS, but some staining with HABP was observed along the borderline between the ameloblasts and the enamel. The significance of HA in the stellate reticulum is discussed. The importance of carrying out investigations with and without protease digestions is stressed.

Immunohistochemistry of the intercellular matrix components and the epithelio-mesenchymal junction of the human tooth germ.

The immunohistochemical localization of heparan sulphate, collagen type I, III and IV, laminin, tenascin, plasma- and cellular fibronectin was studied in tooth germs from human fetuses. The lamina basalis ameloblastica or membrana preformativa, which separates the pre-ameloblasts from the pre-dentin and dentin, contained heparan sulphate, collagen type IV, laminin and fibronectin. Enamel reacted with antifibronectin, but the reaction varied depending on the type of fibronectin and the source of antibody. In early pre-dentin, collagen type I, laminin, tenascin and fibronectin were present. In late pre-dentin and dentin collagen type I was found in intertubular dentin and in the zone between enamel and dentin. The close relationship between collagen type I in dentin and fibronectin in immature enamel is interesting, as it may contribute to the stabilization of the amelodentinal interface. In dental pulp, collagen type IV and laminin were found in the endothelial basement membranes. Collagen type I and III, tenascin and fibronectin were localized to the mesenchymal intercellular matrix. The results of this study have supported the assumption that the lamina basalis ameloblastica is a basement membrane, and have lead to the suggestion that ameloblasts are producers of fibronectin or a fibronectin-like substance.

Quantitative PAS assay of some carbohydrate compounds and detergents.

A spectrophotometric method for determination of color development of glycocompounds subjected to PAS reaction was investigated with various carbohydrate compounds and related chemicals. The conditions of the oxidation with periodic acid was found to influence the amount of the colored Schiff dye produced. Mono- and di-saccharides (mannose, glucose and maltose) were PAS-negative. Glycogen was more reactive than dextran. When glycogen was hydrolyzed by amylase the intensity of the PAS product dropped until a certain limit probably reflecting the limit dextrin. The presence of proteins (albumin) or electrolytes (NaCl) did not influence the PAS reaction. Many non-ionic detergents commonly used in membrane biology such as alkyl glycosides and gluco-methyl alkanamides were strongly PAS-positive and so was the anionic detergent SDS while the zwitterionic detergents tested, such as CHAPS and CHAPSO, were PAS-negative. The color development of the spectrophotometric PAS reaction showed linearity with the concentration of a simple glycoprotein solution (peroxidase) and a complex solution (bovine serum). By the PAS reaction it was also possible to measure the content of soluble and membrane bound carbohydrate compounds in a pellet of liver cell membranes. We find that the PAS reaction is sensitive and reliable for quantitative estimations of complex carbohydrates as well as soluble and membrane-bound carbohydrate compounds. The latter should be treated with PAS-unreactive zwitterionic detergents.

Ultrastructural and morphometric study of hepatocytes from near-term minipig fetuses exposed to ethanol in vivo.

Hepatocytes of near-term minipig fetuses were studied after their mothers had received a daily addition of 3 g/kg body weight of ethanol to the ordinary sufficient fodder during the last half of pregnancy. The ethanol-exposed hepatocytes were evaluated ultrastructurally and morphometrically, and compared with hepatocytes of unexposed fetuses. The present results show that hepatocytes of near-term fetuses, exposed to a high concentration of ethanol for a long period exhibit neither qualitative nor quantitative changes. This is in contrast to maternal hepatocytes which show an adaptation of cellular components to ethanol by developing increased volume densities of mitochondria and smooth endoplasmic reticulum and a decreased volume density of glycogen.

Changes of soluble glycoproteins in dystrophic (dy/dy) mouse muscle shown by lectin binding.

Lectin binding sites in skeletal muscle from normal and dystrophic (dy/dy) C57 BL/6J mice were demonstrated by use of histochemistry and electrophoresis combined with electron microscopy. The following lectins were used: Canavalia ensiformis Con A, Triticum vulgaris (WGA), Glycine max (SBA), Griffonia simplicifolia (GS II), Arachis hypogaea (PNA), Pisum sativum (PSA) and Lens culinaris (LCA). After incubation of frozen sections with Con A, WGA, GS II, PSA and LCA a sarcoplasmic staining was observed in both normal and dystrophic muscle. The most consistent light microscopic observations in the dystrophic muscles were a decreased staining intensity of the sarcoplasm after incubation with Con A, WGA, PSA and LCA, but not with GS II, and a strong staining of the interfiber connective tissue. Supernatants, deprived of organelles and membranes, were prepared from normal and dystrophic muscle by high speed centrifugation. Lectin stained Western blots of the supernatant from dystrophic muscle showed two bands (120 and 67 K) with high affinities to avidin. Further this supernatant contained two glycoprotein bands (180 and 140 K) with affinities to Con A and a number of glycoprotein bands with apparent molecular weights below 67 K showing affinities to LCA and PSA. None of these glycoprotein bands could be detected in the supernatant from normal muscle. These changes of the muscle carbohydrate components might be involved in the expression of the dystrophic syndrome This seems to be the first report on changes of soluble glycoproteins in muscular dystrophy.

Transmission electron microscope observations on enamel following silver methenamine staining.

Transmission electron microscope observations on porcine enamel and secretory ameloblasts showed that silver methenamine material was located inside secretory vesicles in secretory ameloblasts and along the enamel crystals inside immature enamel. It is concluded that silver methenamine is able to stain enamel proteins selectively inside these tissues.

Ultrastructural changes of liver parenchyma following digitonin-pulse perfusion of rat liver.

It has been shown that pulse perfusion of rat liver with a digitonin-containing medium results in a highly zonated hepatocyte permeabilization, allowing selective sampling of cytosolic constituents from periportal and perivenous (centrolobular) hepatocytes "in situ". In the present paper we provide an ultrastructural evaluation of the perfusion method. Identical changes in hepatocytes from affected periportal and perivenous zones are found. Affected hepatocytes appear light (electron-lucent) in electron micrographs with a sharp transition to normal hepatocytes. The most conspicuous ultrastructural findings are: (1) transformation of the sinusoidal part of the light hepatocytes, the lipocyte processes and the endothelium of affected zones apparently unifying into a continuous layer dominated by disrupted plasma membranes and 7-nm filaments; (2) deposition of osmiophilic digitonin-cholesterol complexes along the sinusoidal plasma membranes of affected zones; and (3) reduction of the cytoplasmic matrix (cytosol) in the light hepatocytes, a dilation of the mitochondrial intermembrane space with a preserved mitochondrial matrix, and a dilation of cisternae of the granular endoplasmic reticulum. The ultrastructural findings are consistent with marker-enzyme activity measured in eluates from digitonin-perfused livers, except that lysosomes appear intact, apparently contrasting with the observed eluation of amyloglucosidase (Quistorff et al. 1985).

Scanning electron microscope observations on collagen fibers in human dentin and pulp.

Human permanent teeth were examined in the scanning electron microscope after demineralization and exposure to preparative procedures based on hydrogen peroxide, trypsin, and EDTA. These substances removed the inorganic material, the cellular structures, the homogeneous connective tissue ground substance, and interfibrillar matrix. The remaining tissue components comprised a network of distinct collagen fibers whose organization was related to the type of tissue in which these were incorporated. A similar or identical method has not been developed or applied to teeth previously. Dentin and predentin comprised a compact mass of fibers which basically were parallel to the continuously growing interior surface of the predentin, or arranged at an acute angle to this plane. Collagen fibers in the pulp were numerous, but lacked any particular orientation in most areas. Interodontoblastic fibers crossed the odontoblastic zone at a right angle to the pulp chamber wall and mingled with collagen fibers in predentin. When previously published findings of ours are taken into account, it is possible to conclude that other factors than the organization of the collagen fibers are responsible for the stainability of these fibers in predentin and in interglobular dentin with silver methenamine, and that aldehyde groups on collagen fibers in predentin may be actively and directly involved in the mineralization of the dentin.

Changes of secretory ameloblasts in mini-pig fetuses exposed to ethanol in vivo.

After addition of ethanol to the ordinary fodder of pregnant mini-pigs, ultrastructural changes were found in secretory ameloblasts from tooth germs of their mid-term fetuses. Compared with controls, many mitochondria showed abnormal shape and size, and some exhibited deposition of paracrystalline material in the matrix. An abnormal deposition of stippled material intercellularly was also observed. These changes were interpreted as signs of an abnormal secretory function.

Quantitative study of hepatocytes from minipigs and minipig fetuses exposed to ethanol in vivo.

The effect of ethanol on hepatocytes from pregnant minipigs and their half-term fetuses was studied with the aid of morphometric methods. In the pregnant minipigs the hepatocytes of the ethanol-treated animals showed a significant increase in the volume density of mitochondria, autophagic vacuoles, Golgi complexes and smooth endoplasmic reticulum, and a significant decrease of glycogen. In the half-term fetuses the hepatocytes of ethanol-exposed animals showed no significant change in the volume density of mitochondria, peroxisomes, autophagic vacuoles, Golgi complexes, smooth endoplasmic reticulum or glycogen, and no significant change in the surface density of granular endoplasmic cisternae. The present investigation indicates that in the maternal hepatocyte certain cytoplasmic components are quantitatively changed by ethanol, whereas the volume and surface densities of identical components in the fetal hepatocyte are unaffected. The significance of these results is discussed.

Tetracycline-induced changes in hepatocytes of mini-pigs and mini-pig foetuses as revealed by electron microscopy.

The effect of chlortetracycline on hepatocytes from pregnant mini-pigs and their half-term foetuses was studied after oral administration of 1-3 g daily for 6 days. Liver tissue from tetracycline-treated mini-pigs and their foetuses was immersion fixed, and hepatocytes were evaluated by qualitative and quantitative electron microscopy and compared to hepatocytes of untreated controls. After exposure to tetracycline the hepatocytes of the pregnant mini-pig showed a significant increase in the volume density of mitochondria and autophagic vacuoles. Also a significant decrease in the volume density of glycogen was observed. The hepatocytes of the tetracycline-exposed half-term foetuses showed profound morphological changes of mitochondria. Many mitochondria were of an abnormal shape and an increased size. In most mitochondria the matrix contained a paracrystalline material which had a characteristic prismatic shape with a regular internal lattice structure. Many mitochondria also exhibited numerous short stacked cristae or long longitudinally oriented cristae. No significant change in the volume density of mitochondria was demonstrated. Also no significant change in the volume density of glycogen was observed. In conclusion it can be stated that small doses of chlortetracycline administered orally for a few days provoke morphological changes in both maternal and foetal hepatocytes. While the changes in maternal hepatocytes may reflect metabolic changes, the distinct morphological changes of mitochondria in foetal hepatocytes probably indicate cellular injury.

Electron microscopic demonstration of non-mineralized and hypomineralized areas in dentin and cementum by silver methenamine staining of collagen.

An electron microscopic study on silver methenamine staining of hard dental tissues was made on a material that comprised human permanent teeth and primary tooth germs from human and porcine fetuses. It was demonstrated that silver-stained material consisted of collagen fibrils. In predentin and precementum all collagen fibrils were stained, while collagen fibrils of dentin and cementum were unstained except for some fibrils of minor special areas such as Owen's contour lines, interglobular dentin, Tomes's granular layer, and in cementum small "interglobular-like" areas. It is concluded that silver methenamine visualizes collagen fibrils of hypo- and unmineralized areas in dental hard tissues and therefore may be used to demonstrate abnormal patterns of mineralization. Finally variations of silver methenamine stainability in relation to differences in material and methods were studied and discussed.

Fine structure of hepatocytes from mini-pigs and mini-pig fetuses exposed to alcohol (ethanol) in vivo.

The effect of alcohol on hepatocytes from pregnant mini-pigs and their half-term fetuses was studied after addition of 100-300 g ethanol daily to the ordinary sufficient fodder for about 20 days. Well-defined areas of liver tissue from ethanol-exposed mini-pigs and their fetuses were immersion fixed. The hepatocytes were evaluated ultrastructurally and compared to hepatocytes of non-treated control animals. After exposure to ethanol the hepatocytes of the pregnant mini-pig developed an extensive smooth endoplasmic reticulum, and showed an increased number of mitochondria, microbodies and autophagic vacuoles, extensive Golgi complexes with accumulation of secretion, and a reduction of glycogen. The hepatocytes of the half-term fetuses exhibited profound changes of mitochondria and endoplasmic reticulum after alcohol exposure. Many mitochondria showed abnormal shape and increased size, disorientation of cristae and accumulation of paracrystalline material. An increased number of autophagic vacuoles containing remnants of mitochondria were observed. The granular endoplasmic reticulum exhibited aggregations of endoplasmic cisternae which were well defined and not bounded by a membrane. Thus, the ultrastructural changes in the hepatocytes of the pregnant mini-pig seem to indicate an adaptation of these cells to ethanol by development of a microsomal or catalase ethanol-oxidizing system, while the hepatocytes of the mini-pig fetus in contrast show obvious signs of cellular injury.

Ethanol-induced changes of granular endoplasmic reticulum in hepatocytes of mini-pig foetuses.

The present study describes the occurrence of oblong or oval membranous aggregations of the endoplasmic reticulum in the cytoplasm of hepatocytes from half-term mini-pig foetuses, whose mothers in addition to their ordinary sufficient fodder have been given ethanol in amounts comparable to those consumed by human alcoholics. The structure of the aggregations and their relation to the granular endoplasmic reticulum indicate that they represent a change of the granular endoplasmic reticulum, induced by ethanol. The oval aggregations show resemblances to concentric lamellar bodies described in hepatocytes altered by disease or by experimental procedures not involving ethanol. It is concluded that the change of granular endoplasmic reticulum is probably a sign of an injurious effect of ethanol on the foetal hepatocyte.

Alcohol-induced injury of mitochondria in hepatocytes of mini-pig fetuses.

The present study describes the occurrence of abnormal mitochondria in the cytoplasm of hepatocytes from half-term mini-pig fetuses, whose mothers in addition to their ordinary fodder have received alcohol (ethanol) in daily amounts comparable to those consumed by human alcoholics. The abnormal mitochondria exhibit elongation and distortion, disorientation of cristae and the appearance of paracrystalline and electron-dense material in the matrix. These ultrastructural changes show striking similarities to the mitochondrial injuries seen initially in the hepatocytes of human alcoholics and probably reflect damage to the mitochondria caused by alcohol itself.

Distribution of blood group antigens A, B and H in human fetal oral mucosal and odontogenic epithelium.

The distribution of blood group antigens A, B and H was examined in human fetal oral mucosal and odontogenic epithelium. Tissue from 19 fetuses with crown-rump lengths of 57 mm to 189 mm, corresponding to a fertilization age of 10-20 weeks, was included in the study. The distribution of blood group antigens was studied by immunofluorescence methods on tissue sections. Cell membrane bound blood group antigen A or B was demonstrated in the oral mucosal epithelium of 10 fetuses whereas blood group antigen H was found in all fetuses. All the epithelial cell layers of the tooth germs were devoid of the blood group antigens A, b and H independent of the age of the fetus. The blood group antigens A, B and H were located on the cell membranes of the upper cell layers of the oral epithelium, whereas the basal cells showed no reaction even in the youngest fetuses. Following the differentiation of the epithelial cells, changes were seen in the distribution of the blood group antigens.

Swelling of mitochondria in immersion-fixed liver tissue. Effect of various fixatives and of delayed fixation.

In order to study the occurrence of swollen and disrupted mitochondria in tissue preserved for electron microscopy by ordinary fixation methods, liver tissue from miniature pig fetuses was immersion-fixed in fixatives with various types and concentrations of fixing agents and vehicles. Also commercial and purified products have been tested, different fixation times and temperatures as well as the consequences of a short rinsing in buffer solutions prior to fixation. Furthermore, the significance of delayed fixation (autolysis) was studied. It was found that swollen and disrupted mitochondria occur predominantly in liver cells exposed to low concentrations of glutaraldehyde. It is shown that this phenomenon is a result of a specific effect of glutaraldehyde on the mitochondrial membranes. It is not accompanied by parallel changes of other organelles or nuclei, and it not provoked by other fixing agents, vehicles or by delayed fixation (autolysis).

Morphology of a simple ameloblastoma related to the human enamel organ.

The ultrastructure of a simple ameloblastoma was investigated. The epithelial cells of the tumor could be divided into peripheral cells of varying height, and dark and light central cells. The morphology was correlated to that of the human enamel organ. The low peripheral cells were very similar to the external enamel epithelium cells. The central cells had a certain resemblance to the stellate reticulum and stratum intermedium cells. The high peripheral cells had no counterparts in the enamel organ. Unlike the enamel organ the ameloblastoma showed extremely few and small gap junctions.

Ultrastructure of the human enamel organ. I. External enamel epithelium, stellate reticulum, and stratum intermedium.

The fine structure of external enamel epithelium, stellate reticulum and stratum intermedium in primary tooth germs (bell stage) from four human foetuses was investigated. Characteristically, the cells of the differentiated external enamel epithelium, stellate reticulum and stratum intermedium exhibit many free ribosomes, few rough endoplasmic reticulum cisterns, well-developed Golgi complexes, many coated and smooth vesicles, often in relation to the cell membranes, and many bundles of tonofilaments. The cells are connected by numerous desmosomes and gap junctions. A parallel differentiation of stratum intermedium - external enamel epithelium, and the ameloblast layer is demonstrated. The morphology of the cells of the three layers indicates that these have secretory, transport and supporting functions.

Ultrastructure of the human enamel organ. II. Internal enamel epithelium, preameloblasts, and secretory ameloblasts.

The fine structure of internal enamel epithelium, preameloblasts and secretory ameloblasts in primary tooth germs (bell stage) from four human foetuses was investigated. The characteristics of the differentiation of internal enamel epithelium via preameloblasts to secretory ameloblasts are described. The internal enamel epithelium consists of a row of low differentiated prismatic cells separated from the dental papilla by a distinct even basal lamina. In the preameloblasts the rough endoplasmic reticulum cisterns and mitochondria increase in number, the Golgi complexes become extensive and take up a distal position, and secretory granules are formed. Furthermore, the basal lamina is removed by coated vesicles, and proximally and distally in the cells a complex of zonulae adhaerentes, terminal webs and gap junctions is formed. The secretory ameloblasts make up a layer of highly differentiated cells demonstrating typical merocrine secretion.