Shinji Shimoda

The Zinc Transporter SLC39A13/ZIP13 Is Required for Connective Tissue Development; Its Involvement in BMP/TGF-β Signaling Pathways

Background Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. Methodology/Principal Findings Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-β signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. Conclusions/Significance Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-β signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-β signaling and connective tissue dysfunction.

The Zinc Transporter SLC39A14/ZIP14 Controls G-Protein Coupled Receptor-Mediated Signaling Required for Systemic Growth

Aberrant zinc (Zn) homeostasis is associated with abnormal control of mammalian growth, although the molecular mechanisms of Zns roles in regulating systemic growth remain to be clarified. Here we report that the cell membrane-localized Zn transporter SLC39A14 controls G-protein coupled receptor (GPCR)-mediated signaling. Mice lacking Slc39a14 (Slc39a14-KO mice) exhibit growth retardation and impaired gluconeogenesis, which are attributable to disrupted GPCR signaling in the growth plate, pituitary gland, and liver. The decreased signaling is a consequence of the reduced basal level of cyclic adenosine monophosphate (cAMP) caused by increased phosphodiesterase (PDE) activity in Slc39a14-KO cells. We conclude that SLC39A14 facilitates GPCR-mediated cAMP-CREB signaling by suppressing the basal PDE activity, and that this is one mechanism for Zns involvement in systemic growth processes. Our data highlight SLC39A14 as an important novel player in GPCR-mediated signaling. In addition, the Slc39a14-KO mice may be useful for studying the GPCR-associated regulation of mammalian systemic growth.

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.

Effect of Solution Composition on Morphological and Structural Features of Carbonated Calcium Apatites

The composition of enamel mineral corresponds to that of a calcium carbonato-apatite. For insight to be gained into the precipitation of carbonato-apatites having specific properties (crystal size, morphology, and carbonate incorporation into the crystal lattice), apatites were prepared at 80°C in aqueous systems having various CO3 concentrations and pH values of around 7.5 or 10.5 (± 0.5). The various preparations had a wide range (0.005 to 0.19) of CO3/Ca molar ratios that bracket the ratios found in porcine enamel mineral at various developmental stages. Fourier transform infrared spectroscopy (FTIR) and x-ray diffraction analyses showed that the calcium apatites precipitating at neutral pH incorporated the carbonate into both the hydroxyl and phosphate ion sites in their lattices (A,B-types), whereas the preparations made at the alkaline pH (high OH-CO3 2- competition) or in the presence of fluoride (F--CO3 2- competition) yielded only the B-type carbonato-apatite. It was also ascertained that the size and morphology of the carbonato-apatites, assessed by specific surface area determination and high-resolution electron microscopy, were highly dependent on the driving force for precipitation and the presence of regulators (CO 3 2- and F-) in solution. In neutral media, early precipitates were thin-ribbon in appearance, but grew into crystals having flattened-hexagonal cross-sections. In the presence of fluoride or in alkaline media, acicular apatite crystals, precipitated initially, grew into large rod-like carbonato-apatites having a symmetric-hexagonal cross-section. In both neutral and alkaline solutions, carbonate inhibited the growth of apatite crystals along their c axis, leading to the formation of bulkier crystals. The formation of carbonato-apatites at the neutral pH and their properties are consistent with observations made on enamel minerals formed in the early developmental stages.

Characteristics of Accessory Mental Foramina Observed on Limited Cone-beam Computed Tomography Images

In this retrospective study with limited cone-beam computed tomography (limited CBCT), we investigated the anatomic characteristics of the accessory mental foramina and accessory branches of the mandibular canal. The CBCT records of approximately 150 patients were evaluated, and 17 accessory mental foramina were found in 16 patients. The anatomic peculiarities of the mandibular canal that might be relevant to endodontic treatment were observed. Accessory mental foramina tended to exist in the apical area of the first molar and posterior or inferior area of the mental foramen. The accessory branches of the mandibular canal showed common characteristics in the course of gently sloping posterosuperior direction in the buccal surface area. Verification of the existence of accessory mental foramina would prevent accessory nerve injury during periapical surgery. In root canal treatment, the possibility of accessory mental foramina-related nerve paresthesia seems low unless the mental foramen and mandibular canal are injured. Limited CBCT is effective for presurgical 3-dimensional assessment of the neurovascular structures in dentoalveolar treatment.

Accuracy of intraoral radiography, multidetector helical CT, and limited cone-beam CT for the detection of horizontal tooth root fracture.

OBJECTIVE The accuracies of intraoral radiography (IOR), multidetector helical computerized tomography (MDHCT) at slice thicknesses 0.63 mm and 1.25 mm, and limited cone-beam computerized tomography (LCBCT) were compared for detection of horizontal tooth root fracture. STUDY DESIGN In 7 beagle dogs, 28 maxillary anterior teeth were used, of which 13 had artificially induced horizontal root fracture. The specimens were examined by the above-mentioned 4 modalities. Diagnosis of root fracture was based on direct visualization of radiolucent line in each image by 6 radiologists. RESULTS Sensitivity, negative predictive value, and diagnostic accuracy (true positives + true negatives) for detecting fracture lines in LCBCT (0.96 +/- 0.04, 0.97 +/- 0.03, 0.93 +/- 0.04, respectively) were significantly higher than MDHCT at 0.63 mm (0.76 +/- 0.09, 0.8 +/- 0.05, 0.8 +/- 0.05, respectively), MDHCT at 1.25 mm (0.49 +/- 0.09, 0.66 +/- 0.04, 0.69 +/- 0.05, respectively), and IOR (0.51 +/- 0.18, 0.67 +/- 0.08, 0.69 +/- 0.08, respectively). Specificity and positive predictive value showed no significant intermethod difference among the 4 modalities. CONCLUSION Limited cone-beam CT is more useful than the other 3 radiographic modalities for diagnostic imaging of horizontal tooth root fracture.

Anatomical characteristics of the mandibular lingual foramina observed on limited cone‐beam CT images

OBJECTIVES The study aimed to investigate the regional frequency and anatomical properties of mandibular lingual foramina. MATERIAL AND METHODS The regional frequency of the lingual foramina was investigated using limited cone-beam computed tomographic images of mandibles obtained from 190 examinations of 181 patients. The mesio-distal position of the lingual foramina was classified into 17 areas of the midline area and each tooth area. The vertical position was assessed from the positional relationship between the lingual foramina and mandibular canals. Additionally, anastomoses of the lingual canals from the lingual foramina to mandibular canals were observed on the images. RESULTS One hundred and fifty-four lingual foramina were observed. The highest regional frequency was recognized in the midline area followed by second premolar and canine area. Plural lingual foramina were observed only in the midline areas. No lingual foramina were observed in the superior area of the mandibular canals. Thirty-one lingual foramina showed anastomoses between the lingual canals and mandibular canals. CONCLUSION Information on the high regional frequency of lingual foramina in the second premolar area would be clinically significant to avoid life-threatening incidents because of the possible presence of an artery. It is therefore important to confirm the location of lingual foramina.

Micelle Structure of Amelogenin in Porcine Secretory Enamel

Even during the secretory stage of amelogenesis, enamel crystals thicken as amelogenins (the major protein component) decrease. To explain this phenomenon, we propose a model for amelogenin structure and function based upon the hypothesis that amelogenin forms micelles. Solubility and hydrophobicity analyses suggest that all but the hydrophilic amelogenin C-terminal regions aggregate via hydrophobic bonds to form a micelle core. Amelogenin micelles may form super-assemblies via their C-termini (KTKREEVD), which contain complementary positive (KTKR) and negative (EEVD) elements. Disassembly of the micelles through controlled proteolysis provides space for crystal growth. Initial cleavage (by enamelysin) removes the surface-accessible amelogenin C-terminus, exposing the middle portion to cleavage (by EMSP1). As a result, the 13-kDa amelogenin, a rod-shaped domain based upon ultrafiltration and transmission electron microscopy studies, is released. This model explains how amelogenin is able to ‘space’ and support the ribbon-like crystals and continuously yield space as the crystals thicken, until they are sufficiently mature to support themselves. Abbreviations: dentino-enamel junction (DEJ), sodium dodecyl sulfate (SDS), polyacrylamide gel electro phoresis (PAGE), transmission electron microscope (TEM).

Accuracy of oral mucosal thickness measurements using spiral computed tomography.

BACKGROUND Assessment of oral mucosal thickness is important in implant surgery; however, examining the soft tissue three dimensionally is difficult. A reamer method is invasive, and a non-invasive ultrasonic method produces only low-resolution images depending on anatomic variations. The emerging technology of spiral computed tomography (CT) is an alternative to the conventional methods. Spiral CT has been a useful diagnostic tool in implant surgery. Although it delivers high radiation doses, spiral CT provides three-dimensional imaging of low-contrast structures. The purpose of the present study is to assess the accuracy of oral mucosal measurements using spiral CT. METHODS Thickness of maxillary oral mucosa was measured in five cadavers. The measurement sites were set up in buccal, palatal, and middle of the crest in the missing tooth area in the incisor, canine, premolar, and molar regions. Each cadaver was exposed to spiral CT after installing the measurement guide. After that, each site was physically measured by reamer. Linear regression and correlation analysis were performed to describe the association between radiographic and physical measurements. RESULTS A total of 114 measurements were performed with statistical analyses. Mean values and standard deviations of physical and radiographic measurements were 3.12 ± 1.43 and 2.83 ± 1.70 mm, respectively. The radiographic and physical measurements demonstrate strong correlation (r = 0.90; P <0.01). Measurement error was 0.52 ± 0.36 mm. According to the regions, the measurements in buccal, palatal, and missing tooth region depicted a significant correlation (r = 0.92, r = 0.85, and r = 0.91, respectively). The boundary of the bone and mucosa was indistinguishable at 23 buccal sites. Twenty-three measurements that could not be distinguished with CT had a mean of 0.69 and standard deviation ± 0.13 mm. CONCLUSIONS The correlation between spiral CT and physical measurement was high except in sites of very thin mucosa. Spiral CT can be considered an alternative method for the measurement of oral mucosal thickness. Because of the higher radiation exposure, caution should be exercised and radiation dosage versus clinical benefit assessment is required.

Changes in Acid-phosphate Content in Enamel Mineral during Porcine Amelogenesis

The present study was undertaken to investigate changes in the acid-phosphate content of porcine enamel mineral during its development and to assess separately the HPO4 2- pools in labile and stable forms. Enamel samples at the secretory and maturing stages of amelogenesis were obtained from the permanent incisors of five- to six-month-old slaughtered piglets. Human enamel from erupted, extracted teeth, synthetic hydroxyapatite, and carbonatoapatite containing acid phosphate were included as references. The acid-phosphate content of each sample was determined chemically through its pyrolytic conversion to pyrophosphate. The assessment of HPO4 2- in labile forms was made by analysis of samples pre-equilibrated with solutions containing 3 mmol/L phosphate at pH 11 1 (to de-protonate the HPO4 2- species on crystal surfaces). The analytical results of porcine enamel samples showed that: (a) the outermost secretory (youngest) enamel contained the highest HPO4 2-, corresponding to about 16% of the total phosphate; (b) the acid-phosphate content decreased gradually to 10% in the inner (older) secretory and to 6% in the maturing tissue; (c) a substantial part of the HPO4 2- in developing enamel tissue (50-60% of the HPO4 2- for the secretory enamel) was in labile forms; and (d) the pool of the labile HPO4 2- decreased with the growth of enamel mineral. In parallel studies with mature human enamel, it was ascertained that the total acid phosphate was only about 3% of the total phosphate, much lower than in developing porcine enamel, and that the labile pool of HPO4 2- was also small, corresponding to about 15% of the total acid phosphate determined. The overall results indicate that acid phosphate is one of the major constituents of the early enamel crystals formed during amelogenesis and that the surfaces of the growing enamel crystallites are rich in HPO4 2-.