Takaaki Aoba

Dental Fluorosis: Chemistry and Biology

This review aims at discussing the pathogenesis of enamel fluorosis in relation to a putative linkage among ameloblastic activities, secreted enamel matrix proteins and multiple proteases, growing enamel crystals, and fluid composition, including calcium and fluoride ions. Fluoride is the most important caries-preventive agent in dentistry. In the last two decades, increasing fluoride exposure in various forms and vehicles is most likely the explanation for an increase in the prevalence of mild-to-moderate forms of dental fluorosis in many communities, not the least in those in which controlled water fluoridation has been established. The effects of fluoride on enamel formation causing dental fluorosis in man are cumulative, rather than requiring a specific threshold dose, depending on the total fluoride intake from all sources and the duration of fluoride exposure. Enamel mineralization is highly sensitive to free fluoride ions, which uniquely promote the hydrolysis of acidic precursors such as octacalcium phosphate and precipitation of fluoridated apatite crystals. Once fluoride is incorporated into enamel crystals, the ion likely affects the subsequent mineralization process by reducing the solubility of the mineral and thereby modulating the ionic composition in the fluid surrounding the mineral. In the light of evidence obtained in human and animal studies, it is now most likely that enamel hypomineralization in fluorotic teeth is due predominantly to the aberrant effects of excess fluoride on the rates at which matrix proteins break down and/or the rates at which the by-products from this degradation are withdrawn from the maturing enamel. Any interference with enamel matrix removal could yield retarding effects on the accompanying crystal growth through the maturation stages, resulting in different magnitudes of enamel porosity at the time of tooth eruption. Currently, there is no direct proof that fluoride at micromolar levels affects proliferation and differentiation of enamel organ cells. Fluoride does not seem to affect the production and secretion of enamel matrix proteins and proteases within the dose range causing dental fluorosis in man. Most likely, the fluoride uptake interferes, indirectly, with the protease activities by decreasing free Ca(2+) concentration in the mineralizing milieu. The Ca(2+)-mediated regulation of protease activities is consistent with the in situ observations that (a) enzymatic cleavages of the amelogenins take place only at slow rates through the secretory phase with the limited calcium transport and that, (b) under normal amelogenesis, the amelogenin degradation appears to be accelerated during the transitional and early maturation stages with the increased calcium transport. Since the predominant cariostatic effect of fluoride is not due to its uptake by the enamel during tooth development, it is possible to obtain extensive caries reduction without a concomitant risk of dental fluorosis. Further efforts and research are needed to settle the currently uncertain issues, e.g., the incidence, prevalence, and causes of dental or skeletal fluorosis in relation to all sources of fluoride and the appropriate dose levels and timing of fluoride exposure for prevention and control of dental fluorosis and caries.

Identification of mesenchymal stem cell (MSC)‐transcription factors by microarray and knockdown analyses, and signature molecule‐marked MSC in bone marrow by immunohistochemistry

Although ex vivo expanded mesenchymal stem cells (MSC) have been used in numerous studies, the molecular signature and in vivo distribution status of MSC remain unknown. To address this matter, we identified numerous human MSC‐characteristic genes—including nine transcription factor genes —using DNA microarray and real‐time RT‐PCR analyses: Most of the MSC‐characteristic genes were down‐regulated 24 h after incubation with osteogenesis‐, chondrogenesis‐ or adipogenesis‐induction medium, or 48–72 h after knockdown of the nine transcription factors. Furthermore, knockdowns of ETV1, ETV5, FOXP1, GATA6, HMGA2, SIM2 or SOX11 suppressed the self‐renewal capacity of MSC, whereas those of FOXP1, SOX11, ETV1, SIM2 or PRDM16 reduced the osteogenic‐ and/or adipogenic potential. In addition, immunohistochemistry using antibodies for the MSC characteristic molecules—including GATA6, TRPC4, FLG and TGM2—revealed that MSC‐like cells were present near the endosteum and in the interior of bone marrow of adult mice. These findings indicate that MSC synthesize a set of MSC markers in vitro and in vivo, and that MSC‐characteristic transcription factors are involved in MSC stemness regulation.

Ion transporters in secretory and cyclically modulating ameloblasts: a new hypothesis for cellular control of preeruptive enamel maturation

Mature enamel consists of densely packed and highly organized large hydroxyapatite crystals. The molecular machinery responsible for the formation of fully matured enamel is poorly described but appears to involve oscillative pH changes at the enamel surface. We conducted an immunohistochemical investigation of selected transporters and related proteins in the multilayered rat incisor enamel organ. Connexin 43 (Cx-43) is found in papillary cells and ameloblasts, whereas Na(+)-K(+)-ATPase is heavily expressed during maturation in the papillary cell layer only. Given the distribution of Cx-43 channels and Na(+)-K(+)-ATPase, we suggest that ameloblasts and the papillary cell layer act as a functional syncytium. During enamel maturation ameloblasts undergo repetitive cycles of modulation between ruffle-ended (RA) and smooth-ended (SA) ameloblast morphologies. Carbonic anhydrase II and vacuolar H(+)-ATPase are expressed simultaneously at the beginning of the maturation stage in RA cells. The proton pumps are present in the ruffled border of RA and appear to be internalized during the SA stage. Both papillary cells and ameloblasts express plasma membrane acid/base transporters (AE2, NBC, and NHE1). AE2 and NHE1 change position relative to the enamel surface as localization of the tight junctions changes during ameloblast modulation cycles. We suggest that the concerted action of the papillary cell layer and the modulating ameloblasts regulates the enamel microenvironment, resulting in oscillating pH fluctuations. The pH fluctuations at the enamel surface may be required to keep intercrystalline spaces open in the surface layers of the enamel, enabling degraded enamel matrix proteins to be removed while hydroxyapatite crystals grow as a result of influx of calcium and phosphate ions.

Identification of novel ribonucleo-protein complexes from the brain-specific snoRNA MBII-52

Small nucleolar RNAs (snoRNAs) guide nucleotide modifications within ribosomal RNAs or spliceosomal RNAs by base-pairing to complementary regions within their RNA targets. The brain-specific snoRNA MBII-52 lacks such a complementarity to rRNAs or snRNAs, but instead has been reported to target the serotonin receptor 2C pre-mRNA, thereby regulating pre-mRNA editing and/or alternative splicing. To understand how the MBII-52 snoRNA might be involved in these regulatory processes, we isolated the MBII-52 snoRNP from total mouse brain by an antisense RNA affinity purification approach. Surprisingly, by mass spectrometry we identified 17 novel candidates for MBII-52 snoRNA binding proteins, which previously had not been reported to be associated with canonical snoRNAs. Among these, Nucleolin and ELAVL1 proteins were confirmed to independently and directly interact with the MBII-52 snoRNA by coimmunoprecipitation. Our findings suggest that the MBII-52 snoRNA assembles into novel RNA-protein complexes, distinct from canonical snoRNPs.

Disturbed enamel mineralization in a rat incisor model.

Possession of full-thickness hard enamel appears to be one of the indispensable life-saving characteristics of rats. Previous studies by Suga and his colleagues and by others demonstrated that various types of malformation are evoked in continuously erupting rat incisors. In the current report, we directed our effort to oversee various types of enamel malformation caused experimentally in rat incisors. We surveyed the specimens collected by Suga and his colleagues, as well as specimens we obtained. From the results, it is conceivable that perturbation of the programmed sequential events during enamel development is a major factor in the establishment of enamel malformation. Animal studies with either 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) or a multidentate phosphonic acid (EDTPO) confirmed that dentin mineralization provides a certain inductive effect on the secretion of enamel matrix and subsequent enamel crystallization. Our recent studies using anti-microtubular agents led to the conclusion that the acceleration of mineralization in outer enamel is a type of enamel malformation, most likely due to disruption of the cellular regulation of calcium transport under severe toxic regimens. In future work, experimental approaches combining measurements of kinetic factors with static observation of enamel lesions are required before we can gain a comprehensive understanding of the pathogenesis of disturbed enamel mineralization. The kinetic factors to be considered include the rates of tissue apposition and tooth eruption which determine the total volume of tooth substance formed, and the rate of mineral accretion. Furthermore, information as to the composition, crystallinity, solubility, and mechanical properties of enamel defects is needed before we can assess the susceptibility of teeth having those lesions to caries and other physico-chemical attacks in the oral environment.

Effects of human interleukin-18 and interleukin-12 treatment on human lymphocyte engraftment in NOD-scid mouse

NOD/LtSz‐prkdcscid/prkdcscid (non‐obese diabetic‐severe combine immunodeficiency; NOD‐scid) mice grafted with human peripheral blood lymphoid cells have been used as an in vivo humanized mouse model in various studies. However, cytotoxic human T cells are induced in this model during immune responses, which gives misleading results. To assist in grafting of human lymphocytes without the induction of cytotoxic human T cells, we investigated the effects of T helper type 1 (Th1) and Th2 cytokines on human lymphocyte grafting and migration, as well as the production of immunoglobulin deposited in glomeruli and human immunodeficiency virus‐1 (HIV‐1) infection using NOD‐scid mice. Administration of interleukin‐18 (IL‐18) and IL‐12 enhanced the grafting of human CD4+ and CD8+ T cells in the mice, whereas co‐administration prevented grafting due to interferon‐γ‐dependent apoptosis. Immunoglobulin A (IgA) deposits were observed in mice treated with IL‐18 alone, but not in those given phosphate‐buffered saline, IL‐12 alone, or IL‐18 + IL‐12. A high rate of HIV infection was also observed in the IL‐18‐treated group. Together, these results indicate that IL‐18 may be effective for the grafting and migration of CD4+ and CD8+ T cells, except for the induction of apoptosis and regulation of class‐switching IgA. IL‐18‐administered NOD‐scid mice provide a useful small humanized model for the study of HIV infection and IgA nephropathy.

Three-Dimensional Reconstruction of Oral Tongue Squamous Cell Carcinoma at Invasion Front

We conducted three-dimensional (3D) reconstruction of oral tongue squamous cell carcinoma (OTSCC) using serial histological sections to visualize the architecture of invasive tumors. Fourteen OTSCC cases were collected from archival paraffin-embedded specimens. Based on a pathodiagnostic survey of whole cancer lesions, a core tissue specimen (3 mm in diameter) was dissected out from the deep invasion front using a paraffin tissue microarray. Serial sections (4 μm thick) were double immunostained with pan-cytokeratin and Ki67 antibodies and digitized images were acquired using virtual microscopy. For 3D reconstruction, image registration and RGB color segmentation were automated using ImageJ software to avoid operator-dependent subjective errors. Based on the 3D tumor architecture, we classified the mode of invasion into four types: pushing and bulky architecture; trabecular architecture; diffuse spreading; and special forms. Direct visualization and quantitative assessment of the parenchymal-stromal border provide a new dimension in our understanding of OTSCC architecture. These 3D morphometric analyses also ascertained that cell invasion (individually and collectively) occurs at the deep invasive front of the OTSCC. These results demonstrate the advantages of histology-based 3D reconstruction for evaluating tumor architecture and its potential for a wide range of applications.

Immunodetection of osteopontin at sites of resorption in the pulp of rat molars

Osteopontin (OPN) has been proposed to act as a substrate for osteoclast adhesion during bone resorption. The aim of the present study was to examine the presence and distribution of OPN at sites of resorption in traumatized radicular pulp. The upper first molars of 6-week-old male Sprague-Dawley rats were luxated and then repositioned in the original sockets. The animals were sacrificed by intracardiac perfusion at 10 and 14 days after tooth reimplantation. The teeth were decalcified in EDTA and then processed for embedding in paraffin for histochemistry or LR White resin for immunocytochemistry. Odontoclasts were identified by their multinucleated morphology and expression of tartrate-resistant acid phosphatase (TRAP). Osteopontin was immunolocalized using postem-bedding colloidal gold labeling with a chicken egg yolk anti-rat OPN antibody. After reimplantation of the teeth, TRAP-positive cells were present along the pulp dentin wall. Osteopontin was not consistently detected at exposed predentin/dentin surfaces. However, gold particles were often found at the margin of resorption lacunae. Labeling was also seen over the Golgi region and cytoplasmic vesicles of odontoclasts and of neutrophils and fibroblast-like cells. The results suggest that accumulation of OPN at the predentin/dentin surface is not a prerequisite for adhesion of odontoclasts to the wall substance and that recruited odontoclasts produce OPN locally to mediate cell and/or matrix events within the resorption lacuna.

Hertwig's Epithelial Cells and Multi-root Development of Molars in Mice

Abstract Previously, we focused on multi-root development of the upper and lower molars in mice with the aid of three-dimensional (3D) reconstruction technologies. ICR mice, 3 to 28 days old, were used. The upper and lower jaws including the molars were first used for the collection of micro-computed tomography (μCT) data and, consecutively, were processed to prepare paraffin-embedded sagittal serial sections. Timesequential morphogenesis of the upper and lower first molars was monitored using, μCT-3D models, edited in motion view by superimposing 3D models made at different ages. Histology-based 3D reconstruction in conjunction with immunohistochemistry using cytokeratin as a marker of epithelial cells was also emloyed to visualize the Hertwigs epithelial root sheath (HERS)-guided closure of the pulp chamber floor and the latter-stage disintegration and fate of HERS during root development. The results obtained using 3D models verified that the continuous HERS sheet acts as a guide for root canal segregation and, after disintegration with the underlying dentin formation, HERS-derived epithelial cells have diverse fates, including migration into the periodontium and embedding in the cementum. Recent advances in 3D imaging technology allow us to revisit multiple key issues regarding the developmental morphogenesis and pathological entities of teeth and related tissues.

Use of Microfocus X-ray Computer Tomography for 3D-image Construction and Quantitative Morphoanalysis

Abstract Microfocus X-ray computed tomography (μCT) is becoming widely used in various research fields relevant to the oral sciences. This technology, in conjunction with computer-assisted 3D reconstruction and quantitative structural analysis, is most suitable for investigating hard tissues in a non-invasive way. Despite this recognition, it still remains a concern whether the data acquisition process is conducted properly and accurately in order to ensure the reproducibility of 3D imaging and the determination of structural parameters such as bone volume (BV) and surface area (BS). In this report, we aim to provide an overview of the experimental procedures and conditions that are necessary for optimizing the acquisition of gray-scale CT images and their conversion into digital images (TIFF or JPEG) prior to 3D reconstruction and measurements. Towards these objectives, we collected μCT images of a 4-week-old mouse temporomandibular joint using a μCT apparatus (Nittetsu ELEX Co., ELE-SCAN model). Data proceedings and measurements were conducted using software (RATOC System Engineering Co., TRI-BON and TRI-SRF2). Special care was taken in optimizing experimental conditions and variables, e.g., X-ray tube voltage and current, selection of an adequate shiftvalue to compensate the mismatching between the specimen-rotating center and incident X-ray beam, and window level and width for contrast adjustment. The results of our empirical approach showed that careful selection of the experimental conditions and computation variables ensures the high quality of the 3D images and the accuracy and reproducibility of the quantitative measurements. The magnitude of errors associated with each of the determined structural parameters was confirmed to be within 3% of the determinations. Further knowledge and refinement of μCT technology and data acquisition will help to improve our understanding of the architecture and dynamic modeling of hard tissues.