Yasuo Miake

Epitaxial overgrowth of apatite crystals on the thin-ribbon precursor at early stages of porcine enamel mineralization

SummaryThe aim of the present work was to investigate changes in cross-sectional morphologies of enamel crystallites as a function of location in secretory porcine enamel. Enamel tissues were obtained from 5- to 6-month-old slaughtered piglets. For examination by electron microscopy, a portion of the secretory enamel was embedded in resin and ultrathin sections were prepared with a diamond knife. In parallel studies, compositional and structural changes of enamel mineral were assessed by chemical analysis and Fourier transform infrared (FTIR) spectroscopy. For this purpose, two consecutive layers of the outer secretory enamel, each approximately 30 μm thick, were separated from the labial side of permanent incisors. Using high-resolution electron microscopy, early events of enamel crystal growth were characterized as the epitaxial growth of small apatite units on the lateral surfaces of the initially precipitated thin ribbon. These apatite units had regular triangle or trapezoid cross-sections. After fusions of those isolated trapezoids on both lateral sides of the platy template, the resulting enamel crystallites had the well-documented flattened-hexagonal shapes in cross-sections. The initially precipitated thin plate was buried inside the overgrown apatite lamella and then retained as a central dark line. Similar morphological evidence for the epitaxial nucleation and overgrowth of carbonatoapatite on the platy template was obtainedin vitro. Chemical and FTIR analyses of the enamel layer samples showed that the characteristics of the youngest enamel mineral were distinct from those of enamel crystals found in older secretory enamel. The overall results support the concept that initial enamel mineralization comprises two events: the initial precipitation of thin ribbons and the subsequent epitaxial growth of apatite crystals on the two-dimensional octacalcium phosphate-like precursor.

Functionally graded fluoridated apatites

Fluoridated hydroxyapatite was synthesized at 80 +/- 1 degree C and pH 7.4 +/- 0.2 using a gradient fluoride supply system. X-ray diffraction analysis showed a typically apatitic pattern, although the (3 0 0) reflection was broader than that of homogeneous fluorapatite. Scanning electron micrographic observation indicated that the apatite was composed of rod-like crystals similarly to fluorapatite. High-resolution transmission electron microscopy showed electron damage in the core of the crystal. When the apatite pellet was prepared, electron spectroscopy for chemical analysis showed a negative gradient of fluoride concentration with depth in the crystals. The apparent solubility in 0.5 mol/l acetate buffer solution (37 degrees C and pH 4.0) was 9.16 +/- 0.39 mmol/l, much less than that of homogeneous hydroxyapatite 32.3 +/- 1.9 mmol/l, and less than that of heterogeneous two-layer fluoridated apatite with an outer fluoride-rich layer 12.5 +/- 0.6 mmol/l, which was synthesized previously by supplying fluoride during the latter half of the experimental period. These results suggest that graded fluoridated apatite may be formed by this process and have higher acid resistance than two-layer fluoridated apatite.

Fluoridated apatite synthesized using a multi-step fluoride supply system

Fluoridated apatite was synthesized at 80 +/- 1 degrees C, pH 7.4 +/- 0.2, using a 5-step fluoride supply system. During the synthesis experiment, 0, 5, 10, 15 and 20 mmol/l of fluoride were each supplied for one-fifth of the experimental period with calcium and phosphate. X-ray diffraction analysis showed a typically apatitic pattern, although the (3 0 0) reflection was broader than that of homogeneous fluorapatite. Scanning electron micrographic observation indicated that the apatite was composed of needle-like crystals similar to hydroxyapatite and fluorapatite. High-resolution transmission electron microscopy showed a slender hexagonal shape similar to homogeneous hydroxyapatite in cross-sections perpendicular to the c-axis and the structural damage in the core of the crystal, although no boundary of step-like layers was observed. The apparent solubility in 0.5 mol/l acetate buffer solution (37 degrees C and pH 4.0) was 12.1 +/- 0.2 mmol/l, much less than that of homogeneous hydroxyapatite 32.3 +/- 1.9 mmol/l, and similar to that of heterogeneous two-layer fluoridated apatite with an outer fluoride-rich layer 12.5 +/- 0.6 mmol/l, which was synthesized previously by supplying fluoride during the latter half of the experimental period.

FAM83H and casein kinase I regulate the organization of the keratin cytoskeleton and formation of desmosomes

FAM83H is essential for the formation of dental enamel because a mutation in the FAM83H gene causes amelogenesis imperfecta (AI). We previously reported that the overexpression of FAM83H often occurs and disorganizes the keratin cytoskeleton in colorectal cancer cells. We herein show that FAM83H regulates the organization of the keratin cytoskeleton and maintains the formation of desmosomes in ameloblastoma cells. FAM83H is expressed and localized on keratin filaments in human ameloblastoma cell lines and in mouse ameloblasts and epidermal germinative cells in vivo. FAM83H shows preferential localization to keratin filaments around the nucleus that often extend to cell-cell junctions. Alterations in the function of FAM83H by its overexpression, knockdown, or an AI-causing truncated mutant prevent the proper organization of the keratin cytoskeleton in ameloblastoma cells. Furthermore, the AI-causing mutant prevents desmosomal proteins from being localized to cell-cell junctions. The effects of the AI-causing mutant depend on its binding to and possible inhibition of casein kinase I (CK-1). The suppression of CK-1 by its inhibitor, D4476, disorganizes the keratin cytoskeleton. Our results suggest that AI caused by the FAM83H mutation is mediated by the disorganization of the keratin cytoskeleton and subsequent disruption of desmosomes in ameloblasts.

Ultrastructural study of tissues surrounding replanted teeth and dental implants

OBJECTIVES The aim of this study was to describe the ultrastructure of the dentogingival border at replanted teeth and implants. MATERIAL AND METHODS Wistar rats (8 weeks old) were divided into groups for replantation and implantation experiments. In the former, the upper right first molars were extracted and then immediately replanted. In the latter, pure titanium implants were used. All tissues were fixed, demineralized and embedded in epoxy resin for ultrastructural observations. RESULTS One week after replantation, the junctional epithelium was lost, and the oral sulcular epithelium covered the enamel surface. The amount of the epithelium increased in 2 weeks, and resembled the junctional epithelium, and the internal basal lamina and hemidesmosomes were formed in 4 weeks. One week after implantation, peri-implant epithelium was formed, and in 2 and 4 weeks, this epithelium with aggregated connective tissue cells were observed. In 8 weeks, the peri-implant epithelium receded, and aligned special cells with surrounding elongated fibroblasts and bundles of collagen fibers appeared to seal the implant interface. CONCLUSION In replantation of the tooth, the internal basal lamina remained at the surface of the enamel of the replanted tooth, which is likely to be related to regeneration of the junctional epithelium and the attachment apparatus at the epithelium-tooth interface. Following implantation, a layer of cells with characteristics of connective tissue cells, but no junctional epithelium and attachment apparatus, was formed to seal the site of the implant.

Unusual collagen aggregates induced in rat incisor dentin after vinblastine administration

SummaryYoung male Wistar rats weighing 100 g were injected intravenously with 0.2 mg of vinblastine sulfate (VBL). At 24, 27, and 36 hours and 2, 4, and 6 days after introduction of the drug, the rats were fixed by means of perfusion and the upper incisors were removed for electron microscopy.Many secretion granules had accumulated in the young odontoblasts 24 hours after VBL administration. Newly formed predentin located between odontoblasts contained small amounts of an unusual collagen aggregate characterized by a fine, symmetrically striated structure, and paired, dense bands, which were present at both ends. After 27 hours, following the disappearance of the secretion granules in the odontoblasts, unusual collagen aggregates had increased in amount. Two days after administration, the predentin layer, which was close to the odontoblasts, was composed largely of unusual collagen aggregates. During dentinogenesis, the layer of unusual collagen aggregates gradually shifted into the dentin, where, 4 days after VBL administration, it was, to a large degree, located. Mineralization of the unusual collagen aggregates began with the deposition of fine, filamentous crystals that later grew into needle-shaped crystals 40 Å thick.These findings suggest that unusual collagen aggregates are produced as a result of the secreting function of odontoblasts and are mineralized, as are normal collagen fibrils in dentin.

Immunolocalization of TAK1, TAB1, and p38 in the developing rat molar

In tooth development, transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) are involved in cell differentiation and matrix protein production. TGF-β and BMP have two signaling pathways: the Smad pathway and the non-Smad pathway. However, only a few studies have focused on the non-Smad pathway in tooth development. TGF-β-activated kinase 1 (TAK1) is activated by TGF-β or BMP and binds to TAK1-binding protein (TAB1), activating p38 or c-Jun N-terminal kinase (JNK), forming the non-Smad signaling pathway. In this study, we examined the distribution of these kinases, TGF-β receptor 1 (TGF-β-R1), BMP receptor-1B (BMPR-1B) and Smad4 in cells of the rat molar germ histochemically, in order to investigate the signaling pathway in each type of cell. Immunostaining for TGF-β-R1, BMPR-1B, Smad4, TAK1, TAB1 and phosphorylated-p38 (p-p38) showed similar reactions. In the cervical loop, reactions were clearer than in other enamel epithelium. In the inner enamel epithelium, signal increased with differentiation into ameloblasts, became strongest in the secretory stage, and decreased rapidly in the maturation stage. Signal also increased upon differentiation from preodontoblasts to odontoblasts. In Hertwig’s epithelial sheath, with the exception of BMPR-1B, reactions were stronger in the later stage, showing more enamel protein secretion than in the early stage. However, no clear reaction corresponding to phosphorylated-JNK was observed in any type of cell. These results suggest that TGF-β or BMP is involved in the induction of differentiation of inner enamel epithelium cells into ameloblasts, and preodontoblast differentiation into odontoblasts, the regulation of cervical loop cell proliferation, the elongation or regulation of the epithelial sheath, and the secretion of enamel protein and dentin matrix protein through the non-Smad signaling pathway via TAK1, TAB1 and p38 as well as Smad signaling pathways in the rat molar germ.

Ultrastructural Analyses of Alveolar Bone in a Patient With Osteomyelitis Secondary to Osteopetrosis: A Review of the Literature.

Osteopetrosis is a generic term for generalized sclerotic conditions caused by rare genetic disorders. Decreased osteoclastic activities disturb bone remodeling, resulting in greater mineral density and greater compressive strength; therefore, bone fracture is a major physical symptom of osteopetrosis. Osteomyelitis of the maxilla or mandible is a common and well-documented complication of osteopetrosis. Local infection, such as odontogenic infection, is more likely to lead to osteomyelitis, and treatment strategies can be challenging. However, detailed ultrastructural analyses of bone from patients with osteopetrosis and odontogenic infection are limited. This report describes a case of osteomyelitis of the maxilla and mandible secondary to osteopetrosis in an adult patient and presents ultrastructural data of alveolar bone tissue analyzed by contact microradiography, electron probe microanalysis, and x-ray diffraction. Cases of osteomyelitis of the jaw secondary to osteopetrosis also are reviewed.

Identification, characterization, and expression of dentin matrix protein 1 gene in Xenopus laevis

Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein expressed mainly in bone and dentin, and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. The DMP1 gene, however, appears to evolve rapidly in comparison with other SIBLING genes, even though such functionally important molecules usually evolve more slowly than less important ones. The purpose of this study was to identify and characterize an ortholog of the DMP1 gene in an amphibian (Xenopus laevis; X. laevis) to clarify molecular evolutionary alterations in DMP1 associated with calcified tissues in tetrapods. Furthermore, we analyzed the mRNA expression of this gene to elucidate its functional change in bone and developing tooth germ in comparison with amniote DMP1s. The similarities of the deduced amino acid sequence of X. laevis DMP1 to that of the corresponding amniote proteins were low, although they did share several unique features specific to DMP1 and have similar properties. Expression of X. laevis DMP1 mRNA was predominant in osteocytes and odontoblasts, but only transiently observed in ameloblasts, as in amniotes. These results suggest that DMP1 has conserved several functions during tetrapod evolution. This indicates that continuity of biochemical properties has been more important in maintaining DMP1 functionality than that of the sequence of amino acid residues, which has undergone change over the course of molecular evolution.

The Relationship Between the Structure and Calcification of Dentin and the Role of Melatonin

The present study aimed to examine the relationship between the structure and composition of dentin and odontoblasts and the role of melatonin during the calcification process. The expression of MT1 and MT2 melatonin receptor was confirmed in the odontoblasts of the control group. In addition, the expression of MT1 was stronger than that of MT2. A strong expression of MT1 was detected in the odontoblasts in the melatonin-treated groups. MALDI-TOF MS analysis revealed that two peaks of 795 m/z and 818 m/z were found in dentin. These peaks increased commensurately with the amount of melatonin. The number and size of calcospherites in predentin increased in proportion to the concentration of melatonin. The degree of mineralization increased slightly in the melatonin-treated group using CMR analysis. Two peaks could be clearly detected in the melatonin-treated group at nighttime using X-ray diffraction analysis. Melatonin may participate in the dentin composition and the calcification mechanism of dentin.