Yoshihisa Toda

A scanning electron microscopic observation of etched human peritubular dentine

Abstract Thirty permanent human teeth were fractured immediately after extraction and fixed with 10 per cent neutral formalin or cold acetone. The fractured surfaces were etched with 1 20 N and 1 200 N formic acid for 5–20 and 30–200 sec, coated with gold and the fractured surfaces of the dentine were studied with a scanning electron microscope. In the dentine, intertubular dentine, peritubular dentine and Tomes fibres were distinguishable with these techniques. While the entire peritubular dentine showed a mild resistance to acid treatment, a layer of greater insolubility was also found contiguous upon the dentinal tubules. This probably represents an inner hypomineralized layer.

Ultrastructural cytochemistry of complex carbohydrates in osteoblasts, osteoid, and bone matrix

SummaryGlycosaminoglycans (GAGs) and glycoproteins are essential components for osteogenesis. We have examined rat osteoblasts, osteoid, transitional zone, and fully calcified bone matrix, utilizing Spicers high-iron diaminethiocarbohydrazide-silver protein (HID-TCH-SP) method for sulfated glycoconjugates and Thiérys periodate-TCH-SP (PA-TCH-SP) method for vicinal glycol-containing glycoconjugates. HID-TCH-SP stained cytoplasmic granules of osteoblasts. Stain deposits in the extracellular matrix were observed in decreasing amounts in osteoid, the transitional zone, and fully calcified bone matrix. Enzyme digestion with testicular hyaluronidase removed most HID-TCH-SP stain deposits. PA-TCH-SP staining was observed with increasing intensity in rough endoplasmic reticulum, Golgi saccules, and cytoplasmic granules. Collagen fibrils in osteoid were weakly stained with PA-TCH-SP, and their staining appeared even weaker in fully calcified bone matrix. In contrast, collagen fibrils in calcified cartilage stained intensely with the PA-TCH-SP method. Focal circular profiles (0.1–0.5µm in diameter), which lacked collagen fibrils but reacted moderately with PA-TCH-SP, were frequently seen in the transitional zone and fully calcified bone matrix, but were only occasionally present in osteoid. The presence of testicular hyaluronidase-resistant GAG and acid phosphatase in these focal areas suggests that they represent sites of GAG degradation. The eventual loss of HID-TCH-SP staining in the bone matrix suggests that removal of sulfated glycoconjugates may be a requisite for expansion of initial calcification sites and/or complete calcification.

Ultrastructural visualization of elastic fibres with a tannate-metal salt method

SummaryA modification of the tannic acid-metal salt method was applied as an ultrastructural stain for elastin. Thin sections of glutaraldehyde-fixed, embedded rat aorta and rabbit elastic cartilage, with and without osmication, were examined. Raising the pH of the tannic acid solution from 2.7 to 9.0 progressively increased the electron-density of elastic fibres and collagen fibrils in osmicated and unosmicated specimens. The maximum tannic acid staining of elastic fibres was observed in the pH range 7.0–9.0. Collagen staining, although less intense than that of elastic fibres, was also greatest in this pH range. Elastic fibres in osmicated specimens demonstrated the strongest tannic acid staining with a minimal increase in density of collagen and cell nuclei when compared to the unosmicated specimens. Sequential treatments of osmicated specimens with tannic acid pH 7.0–9.0, and uranyl acetate, pH 4.1, enhanced the density of the elastin intensely, increased collagen staining moderately, but hardly increased the density of nuclei and microfibrils. In elastase-digested osmicated specimens, all tannic acid (pH 7.0)-uranyl acetate-reactive elastin was selectively removed. These results demonstrate that all the neutral and alkaline tannic acid-uranyl acetate methods can be used as a postembedment stain for elastin specimens fixed in glutaraldehyde and osmium tetroxide.

Gene expression of TGF-β1 and elaboration of extracellular matrix using in situ hybridization and EM radioautography during dentinogenesis

The expressions of TGF‐β1 and Type I collagen mRNA were studied by in situ hybridization and immunohistochemistry then the secretory pathway of dentin phosphoprotein was investigated electron microscopic radioautography in rat incisors.

Ultrastructure of Extracellular Polysaccharides Produced by Serotype c Streptococcus mutans

The ultrastructure of extracellular polysaccharides produced in colonies by two clinical isolates and that of a nitrosoguanidine-induced mutant of serotype c Streptococcus mutans with different polysaccharide-synthesizing abilities were compared electron-microscopically. A large amount of polysaccharide was produced from sucrose by colonies of typical serotype c strain MT8148R and a clinical variant MT6801R with an enhanced fructan-synthesizing ability. Transmission electron-microscopy (TEM) revealed that the polysaccharides consisted of three structural components, i.e., globular, single-stranded filamentous, and double-stranded fibrillar structures. These structures were ascribed to production of fructan, water-soluble glucan, and water-insoluble glucan, respectively. On the other hand, two kinds of structures, a globular body and an amorphous substance, were observed by scanning electron-microscopy (SEM). The former was composed of fructan, while the latter contained a mixture of water-soluble and water-insoluble glucans which formed filamentous and double-stranded fibrillar structures under TEM. Very small quantities of polysaccharides were formed in colonies of mutant NG7183, which was derived from S. mutans MT6801R. This strain was found to possess low glucan- and no fructan-synthesizing abilities. The polysaccharides produced in colonies of mutant NG7183 were composed only of filamentous and double-stranded fibrils under TEM. A small amount of amorphous substance was observed by SEM in colonies of NG7183.

Immunoelectron microscopic studies of glycosaminoglycans in the metaphyseal bone trabeculae of growing rats

SummaryThe types and distribution of glycosaminoglycans (GAGs) were studied immunocytochemically in osteoid, mineralized bone matrix, and cartilage matrix of growing rat metaphyseal bone after aldehyde fixation and EDTA demineralization, using four monoclonal antibodies (mAbs 1-B-5, 2-B-6, 3-B-3 and 5-D-4). These mAbs specifically recognize epitopes in non-sulphated chondroitin (C0-S); chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS); chondroitin 6-sulphate (C6-S) and C0-S; and keratan sulphate (KS) respectively. In osteoid, all mAbs except 1-B-5 weakly stained matrix material on and between collagen fibrils, and moderately stained organic material corresponding to bone nodules, which are known sites of mineralization. However, the staining of osteoid abruptly decreased at the mineralization front; weak staining was confined mostly to the organic material of bone nodules in mineralized bone matrix, with very weak or no staining of the rest of the bone matrix. This staining progressively decreased toward the mineralized cartilage matrix and became negative. The mineralized cartilage matrix and lamina limitans reacted strongly with all mAbs except 5-D-4. These results indicate that osteoid contains sulphated proteoglycans containing C4-S and/or DS, C6-S and KS, and subsequent bone matrix mineralization appears to require accumulation of these macromolecules within bone nodules and eventual loss of these substances for complete mineralization, whereas proteoglycans containing C0-S, C4-S and/or DS, and C6-S, still exist in mineralized cartilage matrix and lamina limitants.

Ultrastructural cytochemistry of oxytalan fibres in monkey periodontal ligaments with the high iron diamine method

SummaryMonkey periodontal ligaments have been examined at the ultrastructural level to demonstrate the nature of reactive sites in oxytalan fibres. The high iron diamine (HID) and HID-thiocarbohydrazide-silver proteinate methods specific for sulphate groups, with and without prior oxidation with monopersulphate, were used. Oxytalan fibres were composed of bundles of microfibrils with a diameter of 11.5 ± 1.7 nm (mean ±s.d.,n = 50). In cross section the microfibrils were found to have a denser periphery, giving them a ‘tubular’ appearance. The oxytalan microfibrils of non-oxidized specimens showed little reactivity with either HID method, except that the extracellular matrix material in close association with collagen fibrils stained weakly; in oxidized specimens, both HID methods strongly stained oxytalan microfibrils and weakly stained the extracellular matrix material. Such reactivity of oxytalan microfibrils was not altered by digestion with testicular hyaluronidase or chondroitinase ABC, performed prior to or after persulphate oxidation. Further, the sequential thiosulphation and HID method for the demonstration of disulphide and sulphhydryl groups stained oxytalan fibres moderately. These results indicate that the oxidative generation of sulphate groups in oxytalan fibres may occur from either disulphide or sulphhydryl groups, or both, rather than the result of unmasking of sulphated glycosaminoglycans.

Electron-microscopic observation of adherence of serotype c Streptococcus mutans to the enamel surface due to glucan synthesis

Cellular adherence of three strains with water-insoluble glucan (IG)-synthesizing ability and a strain lacking the ability of serotype c Streptococcus mutans to the saliva-coated human enamel surface was examined by scanning (SEM) and transmission (TEM) electron-microscopy. SEM revealed that organisms of all strains used adhered directly to the enamel surface in the absence of sucrose. Cell-to-cell attachment was scarcely observed in the absence of sucrose. Cell-to-cell attachment via amorphous substance on the cell surface was observed by SEM when the strains with IG-synthesizing ability were incubated with the saliva-coated enamel in the presence of sucrose. TEM revealed that cell-associated enzymes of these strains synthesized filamentous and double-stranded fibrillar structures from sucrose. The strain lacking IG-synthesizing ability was unable to induce cell-to-cell attachment in the presence of sucrose, nor was it able to synthesize the amorphous substance. These results indicate that production of IG by cell-associated glucosyltransferase participates in cellular accumulation of serotype c S. mutans.