Shoichi Suga

Mechanisms and Phylogeny of Mineralization in Biological Systems

Elemental analyses on the growth front of the paper shell obtained from a chambered oyster, Crassostrea gigas, were carried out using the newly developed synchrotron radiation X-ray fluorescence analysis. This paper discusses the possibility of using the synchrotron monochromatized X-ray scanning microbeam to analyze mineralized samples. The newly formed paper shells were analyzed two dimensionally with an x-z scanner. The number of scanning spots was approximately 3000 to 4000. The distribution maps of Ca, Fe, Zn and Sr were obtained. Ca distribution corresponded well with that of membrane-like shell materials and its pattern was similar to that for Sr. Fe and Zn did not represent any characteristic elemental locality in the sample without jelly-like materials except for a rather high accumulation at some small areas. Particular interest is given to Zn, which was similar to the distribution patterns of Ca and Sr in the sample with jelly-like materials. This analytical system is very promising in the field of biomineralization.

Enamel Hypomineralization Viewed From the Pattern of Progressive Mineralization of Human and Monkey Developing Enamel

Microradiograms and their computer-aided image analysis of ground sections of the developing enamel of human permanent third molars and monkey permanent teeth (Macaca fuscata) indicate that the mode of progressive mineralization of enamel is completely different between the matrix formation and maturation stages. During the former stage, the enamel matrix is slightly mineralized. During the latter stage, which takes a much longer period than the previous stage, the increase in the secondary mineralization takes place first slightly, from the surface toward the inner layer, and then heavily, from the inner layer toward the surface. The narrow outer layer mineralizes very slowly during the middle and late stages of maturation, but finally achieves the highest mineralization of the entire enamel layer. The very narrow innermost layer mineralizes slowly without expanding its width. The former three processes seem to be under the direct control of the ameloblasts. Hypoplastic areas which appear during the matrix formation stages are not necessarily accompanied by hypomineralization. Dysfunction of the cells immediately after the completion of matrix formation appears to cause hypomineralization throughout the entire width of matrix except for the innermost layer. Disorders of the cells occurring during the middle and/or the late stage of maturation—due to chronic metabolic disturbances, such as fluorosis—induced hypomineralization localized mainly at the outer layer. The hypomineralized enamel is not necessarily accompanied by hypoplasia. The process of enamel mineralization is not necessarily fully synchronized with that of tooth eruption. Therefore, the narrow outer layer, especially in the fissure and cervical regions, is sometimes hypomineralized even after the teeth have erupted normally.

Ultrastructural studies on crystal growth of enameloid minerals in elasmobranch and teleost fish

SummaryThe composition and morphology of crystals formed in fish enameloid were investigated at various developmental stages. Species studied were shark, skate, red seabream, puffer, and carp. For comparative purposes, mammalian enamel samples were obtained from developing porcine teeth and erupted human teeth. Chemical and physical analyses (FTIR, X-ray diffraction, and electron microprobe) indicated that the mineral phase of enameloid in elasmobranch and teleost fish was most adequately characterized as fluoridated carbonatoapatites but that the degree of fluoridation and carbonation of the apatite latice varied among species and, within species, with the developmental stage. High resolution electron microscopy demonstrated differences in the nature and morphology of the initially precipitating crystallites of the enameloids of elasmobranch as compared with those of teleost fish. The elasmobranch enameloid contained high levels of fluoride (2.5% wt or more) at the beginning of precipitation and its mineralization was characterized by the initial formation and subsequent growth of prismatic apatite crystals having hexagonal (frequently equilateral) cross-sectional areas. In contrast, the initially precipitating crystallites in the teleost fish appeared as thin ribbons, like those commonly reported in mammalian enamel. The crystal morphology of the teleost fish enameloid may be related to the low fluoride contents maintained in the early stages of enameloid formation. However, the growth process of enameloid crystallites varied within the teleostei depending on the fluoride accretion during the mineralization stages. In the seabream enameloid (the highfluoride group), the growth on the side planes of the apatitic prisms was accelerated with increasing fluoride concentration in the tissue; the resulting crystallites had equilateral hexagonal cross sections. The morphology and growth of enameloid crystallites in puffer and carp (the low-fluoride group) were similar to those reported in mammalian enamel. However, appreciable differences existed between the enameloid of this fish group and the mammalian enamel with respect to carbonation, fluoridation, and central defects of their crystallites. The overall results support the contention that fluoride significantly affects the morphology and structure of enameloid crystals, most probably by increasing the driving force for carbonatoapatite formation and by accelerating hydrolysis of possible acidic precursors.

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A comparative study of disturbed mineralization of rat incisor enamel induced by strontium and fluoride administration.

The disturbed pattern of mineralization of developing enamel of the rat incisor after the oral administration of SrCl2 and NaF was investigated in an attempt to disclose possible mechanisms which might not be readily detectable under normal conditions, but which may control the progressive mineralization of developing enamel, especially during the maturation stage. Undemineralized ground sections of upper incisors were examined by contact microradiography, tetracycline labeling, and electron microprobe analysis. It was clear that Sr and F disturb the pattern of mineralization during the maturation stage in a characteristic fashion. Sr inhibits the early stage of maturation in which mineralization progresses from the surface toward the middle layer, whereas F accelerates the same stage prominently. At the late stage of maturation, the pattern of hypomineralization is different in the enamel of Sr- and F-treated rats. Mineralization in the inner and innermost layers of the Sr-treated rats and that in the outer layer of the F-treated rats ceases earlier than that in the controls, although the enamel is still hypomineralized. At the latest stage of maturation, Fe penetrates more deeply into the hypomineralized enamel of the Sr- and F-treated rats, because of the higher porosity of the matrix. These results suggest that the maturation stage is not a simple, continuous process, but rather is composed of substages (phases) which have different control mechanisms and in which mineralization progresses in different modes and rates.

Crystallo-chemical properties of apatite in atremate brachiopod shells.

The shells of atremate brachiopods are unique in that their shells consist of calcium phosphate instead of calcium carbonate [I-4]. Based on x-ray diffraction analyses, the shell mineral of brachiopod Lingula was identified as dahllite, a C03-containing calcium-OH-apatite [1,5]; while based on fluorine (F) content and x-ray diffraction analysis, it was identified as being crystallo-chemically identical with francolite, a CO 3containing calcium-F-apatite [2]. The shell mineral of brachiopod Pel~aiodiscus atlanticus was first identified as dahllite based on x-ray diffraction data alone, and as francolite, based on additional F content data [4]. Infrared absorption analyses to determine the structural involvement of the CO 3 in the brachiopod shell apatitewhether as a component of a separate calcium carbonate phase or as a substltuent in the apatitehas not been used in any of the previous study.

Iron in the Enameloid of Perciform Fish

It is known that a high concentration of iron is deposited in the enameloid of some teleostean fish. Previously, Suga et al. (1989) pointed out that the iron concentration in the enameloid is related to the phylogeny of fish rather than to the feeding habits, according to the results of quantitative iron analyses on the teeth of marine teleost fish of the Tetraodontiformes. In the present study, in order for the previous idea to be verified, quantitative iron analysis was made with an electron microprobe on the enameloid offish belonging to the Perciformes, which is the largest group of teleostean fish in the world and consists of both marine and freshwater species. The enameloid of all the fish examined (57 species) contained high iron concentrations ranging from 0.2% to 10.2% at the surface or middle layer, whereas that of an advanced suborder, Tetraodontoidei, of the Tetraodontiformes was very low in iron, at a level which could not be discriminated from the background value of the emission intensity. The distribution pattern of iron in the enameloid was classified into at least two types, namely, type A, in which a high iron concentration was observed mainly in the surface layer, and type B, in which iron was deposited throughout the entire layer, although there were differences in concentration. There were some differences in the concentration and distribution of iron in the enameloid for the families; for example, those of the Scaridae had a type A distribution, with about 0.2% iron only at the surface layer, whereas those of the Cichlidae, Centrarchidae, and Acanthuridae, which showed a type B distribution, contained iron ranging from 2.9% to 10.5% at the surface or middle layer of enameloid. Such differences seemed to be associated with the difference in timing of the commencement of the iron deposition into the developing enameloid, which is probably related to the phylogeny of fish. There was no evidence to support the idea that the iron concentration in the enameloid is associated with the feeding habits offish, as proposed by previous investigators.

Calcification in Higher Plants with Special Reference to Cystoliths

The cystolith, a calcified body in the leaf of higher plants, was observed by soft X-ray microradiography using the mature leaves of nine species from five families. The microradiographs revealed very large cigar-shaped cystoliths (up to 500 μ m in length) in Pilea viridissima and Justicia procumbens, neighbor-cystoliths in Morus bombycis and Humulus scandens, and two to seven radially arranged cystoliths in Momordica charantia. The number of cystoliths (n/cm2) of various kinds of leaves vas estimated to be 850 to 4,200 by microradiography. The calcium carbonate content (mg/cm2) calculated was 0.3 to 1.1, suggesting a large reservoir of calcium or carbon dioxide. The cystoliths were isolated from leaves of nine species to analyze chemically, with electron probe, and with X-ray diffraction. The calcium carbonate content in cystoliths was about 75% on a dry weight basis. A small amount of magnesium was also found. Electron probe analysis revealed that calcium and magnesium were evenly distributed through the cystolith body except for the stalk and the basal part of the body to which the stalk is attached. In the latter parts, silicon was detected in high density, suggesting silicification of these parts. X-ray diffraction patterns showed amorphous calcium carbonate in all species tested. However, vaterite in Morus bontbycis and both vaterite and calcite in Ficus elastica were also detected in small amounts. The amorphous calcium carbonate in cystoliths changed rapidly into calcite in 0.05 M carbonate buffer (pH 9.2) or in distilled water. Then the smooth surface of the cystoliths was covered with small cubic calcite crystals. A tremendous number of cystoliths was contained in a single leaf, and the cystolith-bearing lithocyst was associated with many photosynthetic parenchyma cells in all species. These facts suggest a relationship between calcification in the cystolith and photosynthesis in the leaf. A possible mechanism of cystolith calcification coupled with bicarbonate utilization in photosynthesis is discussed.

Iron Concentration in Teeth of Tetra-odontiform Fishes and its Phylogenetic Significance

It is known that iron is deposited in the enameloid of some teleost fishes, although its biological significance has not been clarified. In the present investigation, a quantitative analysis of iron in the enameloid of fishes of a primitive suborder, the Balistoidei, and an advanced suborder, the Tetra-odontoidei, of the Tetra-odontiformes of marine teleosts was performed by means of the electron microprobe. The results indicated that the enameloid of Balistoidei contained from 0.4-13.5% iron at its surface layer, whereas that of Tetra-odontoidei was very low in iron, which could not be discriminated from the background value of the emission intensity. The enameloid of three perciform species belonging to the Acanthuridae-from which the Tetra-odontiformes are considered to have been derived-also contained high iron (2. 7 - 3.9%) throughout its entire layer. The iron concentration in the enameloid seemed to be related to the phylogeny of fishes rather than to their environmental water and feeding habits, and it is believed that the mechanisms of iron concentration into the developing enameloid have been lost during evolution from the Achanthuridae to the Tetra-odontoidei. Since a similar phenomenon has been previously observed with respect to the fluoride concentration in the enameloid of the same fishes (Suga et al., 1981a), it is speculated that the concentrations of iron and fluoride, which have originally no chemical correlation, have some special biological significance, although the timing and distribution pattern of their deposition are completely different.

Application of soft X-ray microradiography to observation of cystoliths in the leaves of various higher plants.

Soft X-ray microradiography was applied to observation of the cystoliths, calcified bodies of higher plants, in the leaves ofMorus bombycis, Humulus scandens, Ficus elastica, F. retusa (Moraceae),Boehmeria platanifolia, Pilea viridissima (Urticaceae) andMomordica charantia (Cucurbitaceae). It was proved that this technique is useful for examination of the shape, size, distribution and number of cystoliths in fresh leaves. The microradiographs revealed large cigar-shaped cystoliths in the leaf ofP. viridissima, and neighbor-cystoliths in somewhat restricted areas of the leaves ofM. bombycis andH. scandens, and two to seven radially arranged cystoliths in the leaf ofM. charantia. The number of cystoliths per unit area of leaf (nos./cm2) was estimated to be from 1,090 to 3,900 by means of the microradiographs, varying from species to species. The CaCO3 content of the leaf calculated from the volume and number of cystoliths was approximately 0.4 mg/cm2 in all species exceptF. retusa. InF. retusa, it was about 1.06 mg/cm2, the highest value among all species tested. Hand-sections of the leaves showed that the lithocysts were localized in the upper and/or lower epidermis, and they were associated with many photosynthetic cells in all species, suggesting some relationship between CaCO3 deposition in cystoliths and photosynthesis.