Bending Fibers of Hydroxyapatite for Ordered Parallel Architecture in

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Article Cite This: ACS Omega 2019, 4, 3739−3744

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Bending Fibers of Hydroxyapatite for Ordered Parallel Architecture in Bovine Tooth Enamel Yutaro Yukimasa, Mihiro Takasaki, Yuya Oaki, and Hiroaki Imai* Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan

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S Supporting Information *

ABSTRACT: The nanometer-scale bending structure in the enamel−dentin interfacial region of bovine teeth was clarified using detailed electron micrographs. We found that enamel prisms of width ∼5−10 μm in the enamel are regarded as a bundle of nanometer-scale hydroxyapatite (HA) fibers of width ∼20−30 nm that are connected to the enamel−dentin interface. The radial arrangements of HA fibers elongated in the c-axis were formed on the dentin surface at the boundary. The orientation adjustment of the HA fibers was observed through stepwise bending with a gradual change of the c direction in the ordered parallel array of the enamel prisms. Our observation suggests that the crystallographic ordering in the enamel layer originates from the growth-induced orientation of HA crystals in the interfacial region.

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content from dentin to enamel was shown at the boundary.20 The nanometer-scale structures of dentin, enamel, and the EDJ were reported with their different mechanical properties.8,11 Tiny crystalline phases are randomly arranged with the network of collagen in the dentin, whereas highly oriented HA crystals are formed in the hard enamel. On the other hand, the detailed nanoscale structures of the EDJ layer have been slightly discussed in the previous studies. Whereas the EDJ has been recognized to be more than several micrometers thick (micrometer-scale EDJ),21−23 the intermediate layer was recently reported to be about 200 nm (nanometer-scale EDJ) on the basis of a detailed transmission electron microscopy (TEM) observation (Figure S1 in the Supporting Information).11 Unfortunately, the evolution of the crystallographic orientation was not studied in these works. In the present study, we focused on the detailed structure in the EDJ region of bovine incisors. Bovine teeth have a specific chemical composition and physicochemical properties similar to those of human teeth.24 The enamel structure of incisors was reported to be almost the same as that of other teeth, such as molars.8,11,25 Moreover, bovine teeth are easy to obtain for scientific research. Thus, we used bovine incisors as a typical example for nanoscopic structural observation of the EDJ region. Here, radially arranged HA fibers were found to be the root of the ordered architecture in the interfacial enamel region. The c direction of HA fibers in the prisms is adjusted through stepwise bending for the formation of the parallel array in the enamel prisms. These findings shed light on the

INTRODUCTION Mammalian teeth are covered with hard layers called enamel that are mainly composed of hydroxyapatite (HA, Ca10(PO4)6(OH)2).1 Parallel arrangement of enamel prisms of width ∼3−5 μm is observed in the enamel layers.2 The prisms elongated in the c direction are regarded as a bundle of nanometer-scale HA fibers or rods of width ∼30−40 nm.2−4 The specific mechanical properties of the enamel are ascribed to their hierarchical architecture.5,6 Dentin as a base of the enamel layer is composed of 70 wt % HA, 20 wt % collagen, and 10 wt % water.7 Its elastic nature supports the outermost hard layer of the teeth.8−10 The enamel is basically comprised of three layers: an outer enamel surface (OES), Hunter−Schreger bands (HSBs), and the enamel−dentin junction (EDJ) (Figure S1 in the Supporting Information).11−14 Whereas HA crystals are randomly arranged in the dentin,11 HA fibers are highly oriented in the OES and HSBs of enamel. The movement of an ameloblast, which is a cell that deposits tooth enamel, controls the growth direction of the enamel prism from the surface of dentin.15,16 The cross-lamellar structure in HSBs is fabricated with two kinds of enamel prisms diagonally elongated from the EDJ.17 The enamel prisms are a bundle of nanometer-wide HA fibers that are arranged in the same orientation. Although the elaborate architectures are found to be constructed with curved fibrous structures, their crystallographic structures and formation mechanism have not been revealed through a detailed observation. Clarification of the EDJ region as an intermediary of dentin and enamel18−20 is important to understand the mechanical properties of teeth and the initial formation process of the highly ordered architecture. A gradual change in the organic © 2019 American Chemical Society

Received: January 9, 2019 Accepted: February 6, 2019 Published: February 20, 2019 3739

DOI: 10.1021/acsomega.9b00070 ACS Omega 2019, 4, 3739−3744

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Figure 1. Schematic illustrations (a−c), SEM images (d−i), and EDX mapping (j) of a fractured surface around the EDJ of a bovine incisor. The surface in (g−i) was treated with an EDTA solution.

was found at the boundary between HSBs and PAPs ((I) in Figure 1). The prisms in PAPs were thin at the interface between the PAPs and dentin ((II) in Figure 1). We observed the same structure in the EDJ region of several samples. Figure 2a,b shows enlarged SEM images of a fractured surface of PAPs before and after the EDTA treatment. The prisms are regarded as a bundle of nanometer-scale fibers of width ∼20−30 nm, as mentioned above. The fibrous feature was clarified on the prisms by a mild etching with EDTA, Figure 2c−e shows a TEM image and its selected area electron diffraction (SAED) pattern of a focused ion beam (FIB)-cut plate of PAPs. Bundles of fibers of width ∼30−50 nm are arranged normally to the enamel−dentin boundary. The selected area electron diffraction (SAED) pattern shows that the c direction of HA is roughly normal to the boundary. The deviation of the c direction is ca. 20° in the bundle of HA fibers. On the other hand, diffraction spots are assigned to four sets of zone axes. This means that the a directions of HA fibers are not arranged in the same orientation and rotated around their c-axis. Figure 3 shows enlarged SEM images of the roots of the PAPs near the boundary (II) before and after the EDTA treatment. On the other hand, a detailed structure at the boundary of the enamel prisms and the collagen substrate was revealed after removal of the platelets by the EDTA treatment. The enamel prisms are deduced to be grown from nucleation

ordering mechanism of parallel arrangements of nanometerscale fibers observed in biological mineralization systems.

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RESULTS AND DISCUSSION In the current work, we performed a detailed observation of the EDJ region. Figure 1a−f shows scanning electron microscope (SEM) images and schematic illustrations of a fractured surface around the enamel−dentin interface of a bovine incisor. The presence of calcium phosphate on an organic basal layer was confirmed from the energy-dispersive X-ray spectrometer (EDX) mapping (Figure 1j) and EDX spectrum (Figure S3). Here, we identified two layers in the enamel region near the dentin: a cross-lamellar structure of thickness 20−50 μm, regarded as HSBs, and perpendicularly aligned prisms (PAPs) of thickness 10−20 μm, regarded as a micrometer-scale EDJ. Although the bilayer prisms were observed near the enamel−dentin boundary of a bovine incisor, detailed crystallographic structures have not been characterized in a previous work.24 As mentioned in the Experimental Section, the microscopic structures were revealed by the ethylenediaminetetraacetic acid (EDTA) treatment in the present work. As shown in Figure 1g,h, enamel prisms of width ∼5 μm were clearly observed in the HSBs and micrometer-scale EDJ (=PAPs) on the organic substrate of dentin. Here, the micrometer-scale EDJ is called PAPs due to its structural feature. Distinct bending of continuous prisms 3740

DOI: 10.1021/acsomega.9b00070 ACS Omega 2019, 4, 3739−3744

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Figure 2. Enlarged SEM images of a fractured surface of PAPs before (a) and after (b) the EDTA treatment and a TEM image (c) and its SAED pattern (d) of an FIB-cut plate of PAPs. The diffraction spots in (d) are assigned to four sets of zone axes (e).

to be produced through the ordering of fibers with a gradual change in the growth direction in the enamel−dentin interfacial region. Our findings in the present study shed light on the ordering mechanism of parallel arrangements of fibrous HA crystals observed in the enamels. According to previous works,15,16 nucleation of HA crystals occurs at the interface between dentin and enamel regions. The initial enamel crystals grow from the interface with receding of ameloblasts. Thus, the movement of the ameloblast controls the growth direction of the enamel prism from the dentin surface. This means that the structural analysis around the EDJ of mature teeth provides the information on the developmental mechanism in the initial stage of the enamel formation. The center of radial arrangements of the HA fibers at the boundary indicates the presence of the nucleation site on the dentin surface. The stepwise bending structure suggests that the ordered parallel array of the enamel prisms is produced via orientation

sites on the organic substrate of dentin. Radial arrangements of nanometer-scale fibers are located as the root of the prisms. The direction of the fibers gradually changes, becoming normal to the boundary. These results indicate that HA crystals are nucleated on the collagen surface of dentin and then grown radially in the initial stage of enamel formation. This region is regarded as the nanometer-scale EDJ reported by Chan et al.11 The direction of HA fibers is gradually adjusted to become normal to the surface in the progressive stage. Figures 4 and S4 show TEM images of an FIB-cut plate for observation of the root of the prism in the PAPs. Here, we found bending fibers in the prisms. Parallel planes assigned to (100) in high-resolution TEM images indicate that the fibers are elongated in the c direction. A gradual stepwise change of the c direction is observed on the bending part of a fiber. Thus, low-angle grain boundaries are essential for the bending structures of HA fibers elongated in the c direction. In consequence, the parallel arrays in the enamel layer are inferred 3741

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architectures. A further biological work using dental bulbs is required to clarify the correlation. In our present study, we revealed the crystallographic structure of HA fibers in the enamel prisms. Here, we approached the enamel formation on the basis of the crystallographic structure. The bending structures are deduced to be important for the ordered architecture of the enamel. The bending HA fibers are deduced to be produced by orientation adjustment toward the ameloblast. Simmer et al. showed that various enamel structures were formed with the movement of ameloblasts.27 Thus, the role of the ameloblast is essential for the production of the sophisticated architecture of enamel prisms. The formation mechanism of HA bending fibers was discussed on the basis of the biological growth process of an enamel prism by an ameloblast.15 For instance, the bundled fibers of calcium carbonate crystals are commonly observed in other biominerals, such as cross lamella in seashells and tests of foraminifera.28,29 However, the ordering mechanism for the parallel arrays of the mineral fibers has not been clarified. These findings provide important information for understanding the biological process.

Figure 3. Enlarged SEM images of the roots of the PAPs before (a, b) and after (c, d) the EDTA treatment.

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adjustment of the HA fibers. On the basis of observation in previous works and the present study, we propose a tentative growth process of parallel arrangement in the enamel, as illustrated in Figure 5. An ameloblast is deduced to produce an enamel prism because of the similarity in their widths.15 In the initial stage, each ameloblast induces a nucleus of HA on the collagen substrate of dentin. Tiny crystals form a fibrous shape through elongation in the c-axis. The radial arrangement of HA fibers is produced by crystal growth on the organic substrate.15,26 The growth direction of HA fibers is adjusted to become normal to the interface through stepwise bending with low-angle grain boundaries. On the other hand, the a axes are not arranged in the ordered architecture. Finally, the parallel arrangement of enamel prisms elongated in the c direction is achieved in enamel prisms by the growth-induced orientation of HA fibers. A scalloped structure was observed around EDJ in the previous study.8 However, the period of scallop morphology, which was 20−40 μm, was not close to the size of an enamel prism. Thus, the growth process is not influenced by the scalloped structure. Tomes’ process of ameloblasts and interprisms are generally important for the arrangement of enamel prisms. Here, stepwise bending hydroxyapatite fibers have been found as an important unit in the range of ∼10 μm from the dentin surface of mature teeth. Since the ameloblasts and interprisms are away from the dentin surface in mature enamel,24 we cannot discuss the correlation of the detailed structure of the EDJ and the other

CONCLUSIONS Detailed architectures in the enamel−dentin junction of bovine teeth were investigated to clarify the ordering mechanism for the parallel arrangement of HA crystals in the enamel. Radially arranged HA fibers elongated in the c-axis were found in the interfacial enamel region. A parallel array of the enamel prisms is composed of bending fibers with a gradual stepwise change in the c direction. Therefore, the formation of bending fibers at the enamel−dentin boundary is essential for construction of the highly ordered architecture in bovine tooth enamel.

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EXPERIMENTAL SECTION Bovine incisors of cattle at 20−40 months after birth before eruption stored at −2 °C were used in the present experiment (Figure S1 in the Supporting Information). We fractured the teeth with a hammer and prepared small pieces of the specimen that exposed their cross section for electron microscopies. We used many bovine incisors, more than 20, for observation of the EDJ region. Basically, the same structures were observed in all of the samples. We used 0.1 mol/dm3 ethylenediaminetetraacetic acid (EDTA) solution at pH 7.0 to dissolve tiny HA crystals covering the enamel columns around EDJ.19 Small pieces of the specimen (1−4 mm in size) were immersed in the EDTA solution at room temperature. The detailed microscopic structures were then

Figure 4. TEM images (a−c) of an FIB-cut plate for observation of the root of the perpendicularly aligned prism. Panels (b) and (c) show enlarged images of a square in panels (a) and (b), respectively. 3742

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Figure 5. Schematic illustration of the tentative growth process of HA for parallel arrangement in the enamel. The influence of Tomes’ process on the growth of HA crystals is unclear in the present work.

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revealed by the EDTA treatment because only tiny HA crystals smaller than 10 nm were removed by the mild etching process. A thin-film sample about 100 nm thick was cut from the enamel−dentin boundary using a focused ion beam (FIB) (Figure S2 in the Supporting Information) for field-emission transmission electron microscopy (TEM, FEI Tecnai F20 operated at 200 kV) with selected area electron diffraction (SAED). The observation was also carried out using a fieldemission scanning electron microscope (SEM, Hitachi S-2700, JEOL JSM-7600F) operated at 5.0 kV. Elemental analysis was performed using a Hitachi Miniscope TM 3030 with an energy-dispersive X-ray spectrometer (EDX). The samples were coated with osmium for SEM observation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.9b00070. Photo and SEM images and schematic illustration of cross section of a bovine incisor (Figure S1); SEM images of a FIB-cut surface around the enamel−dentin junction (EDJ) (Figure S2); SEM-EDX spectrum of a tooth sample after EDTA treatment (Figure S3); and TEM images of bending HA fibers in an FIB-cut plate of the EDJ region (Figure S4) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Mihiro Takasaki: 0000-0001-9183-7698 Yuya Oaki: 0000-0001-7387-9237 Hiroaki Imai: 0000-0001-6332-9514 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

Bovine teeth were supplied by Prof. Kunio Ishikawa and Dr Yuki Sugiura at Kyushu University. This work was partially supported by the Grant-in-Aid for Challenging Exploratory Research (15K14129) and Scientific Research (A) (16H02398) from Japan Society for the Promotion of Science. 3743

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DOI: 10.1021/acsomega.9b00070 ACS Omega 2019, 4, 3739−3744