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High Resolution STEM Images of the Human Tooth Enamel Crystals

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High Resolution STEM Images of the Human Tooth Enamel Crystals applied sciences Article High Resolution STEM Images of the Human Tooth Enamel Crystals José Reyes-Gasga 1,* and Etienne F. Brès 2 1 Instituto de Física, Universidad Nacional Autónoma de México, Circuito de la Investigación Científica s/n, Cd. Universitaria, Coyoacán, Ciudad de México 04510, Mexico 2 UMET, UMR 8207 CNRS, Bâtiment C6, Faculté des Sciences et Technologies, Université de Lille, 59650 Villeneuve d’Ascq, France; [email protected] * Correspondence: jreyes@fisica.unam.mx Abstract: High-resolution scanning transmission electron microscopy (STEM) images of human tooth enamel crystals, mainly in the high-angle annular dark-field (STEM-HAADF) mode, are presented in this work along the [1000], [10-11]. and [1-210] directions. These images allow knowing some structural details at the nanometric level of the human tooth enamel crystals and of the central dark line (CDL) observed at their centers. The transmission electron microscopy (TEM) and high- resolution TEM (HRTEM) images of the CDL showed the Fresnel contrast. In the STEM bright-field (STEM-BF) and annular-dark-field (STEM-ADF) images, the CDL was observed as an unstrain hydroxyapatite (HAP)-like zone but surrounded by a strained zone. In the STEM-HAADF images, the CDL appeared with a weak contrast, and its contrasts’ thickness was registered between 3 and 8 Å. The arrangement obtained in the STEM-HAADF images by identifying the bright points with the Ca atoms produced the superposition of the HAP atomic sites, mainly along the [0001] direction. The findings provide further information on the structure details at the center of enamel crystals, which favors the anisotropic carious dissolution at the CDL. Citation: Reyes-Gasga, J.; Brès, E.F. Keywords: human teeth; human tooth enamel; carious tooth enamel dissolution; central dark line; High Resolution STEM Images of the electron transmission and scanning microscopy; annular dark field images Human Tooth Enamel Crystals. Appl. Sci. 2021, 11, 7477. https://doi.org/ 10.3390/app11167477 1. Introduction Academic Editor: Gianrico Spagnuolo 1.1. Human Tooth Structure The human tooth is made up of dentin, which is a connective tissue that gives shape Received: 8 July 2021 and stiffness. In the crown, dentin is covered by enamel, the most wear-resistant tissue in Accepted: 12 August 2021 Published: 14 August 2021 the human body. Enamel is responsible for protecting teeth from wear and tear caused by chewing as well as corrosion from acids produced from food debris. The dentin–enamel Publisher’s Note: MDPI stays neutral junction zone is where dentin meets enamel. with regard to jurisdictional claims in Dentin is made up of a 70% inorganic material, a 20% organic material, and 10% published maps and institutional affil- water. Enamel is composed of a 90% inorganic material, a 5% organic material, and 3% iations. water [1]. The inorganic component is calcium phosphate named hydroxyapatite (HAP, Ca10(PO4)6(OH)2). The organic part is collagen. The EDS analyses of enamel and dentin indicate the existence of substitute elements, such as Mg, Na, and Cl, and residual organic elements, such as C and N, in addition to the HAP elements Ca, P, and O [2,3]. At the micrometric level, enamel is made up of elongated structures arranged in rows Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. with sinuous trajectories from the dentin–enamel junction to the enamel surface named This article is an open access article rods or prisms. This prismatic structure has a keyhole-type shape. At the nanometer level, distributed under the terms and the enamel prisms are made up of ribbon-shaped crystals which are approximately 70 to conditions of the Creative Commons 170 nm long, aligned with the length of the rods. When cross-sectioned, the crystallites Attribution (CC BY) license (https:// appear as semi-polygons with a 20-to-50 nm thickness range [1]. In the center of the enamel 2+ + creativecommons.org/licenses/by/ crystals, small concentrations of Mg and Na have been reported [3], which indicates 4.0/). modifications in the elements of the HAP unit cell at this region. Understanding the Appl. Sci. 2021, 11, 7477. https://doi.org/10.3390/app11167477 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, x FOR PEER REVIEW 2 of 14 Appl. Sci. 2021, 11, 7477 2 of 13 been reported [3], which indicates modifications in the elements of the HAP unit cell at this region. Understanding the structure and chemistry of enamel at the nanoscale is es- structuresential for and elucidating chemistry the of enamel enamel at dissolution the nanoscale process is essential and for for developing elucidating thenew enamel dental dissolutiontreatments. process and for developing new dental treatments. 1.2.1.2. TheThe CentralCentral DarkDark LineLine AtAt thethe centralcentral regionregion ofof thethe humanhuman toothtooth enamelenamel crystals,crystals, aa defectdefect namednamed asas “the“the centralcentral darkdark line” line” (CDL) (CDL) was was observed. observed. In In transmission transmission electron electron microscopy microscopy (TEM) (TEM) images, im- theages, CDL the showedCDL show a contrasted a contrast similar similar to the to “Fresnel the “Fresnel contrast” contrast of” edge-on of edge- defects;on defects; that that is, itis, appeared it appeared as aas dark a dark line line with with a widtha width of of ~1 ~1 nm nm out-of-focus out-of-focus in in both both cross-sectional cross-sectional andand transversetransverse sectionsection samples,samples, butbut itit waswas whitewhite inin over-focusover-focusand anddisappeared disappearedin-focus in-focus (the(the GaussianGaussian focus)focus) [[33––55].]. Figure1 1 shows shows a a bright-field bright-field TEM TEM image image of of human human tooth tooth enamelenamel crystals,crystals, wherewhere thethe arrowsarrows indicateindicate thethe positionspositions ofof thethe CDL.CDL. TheThe insetinset showsshows aa high-resolutionhigh-resolution TEMTEM (HRTEM)(HRTEM)image imageof of the the CDL CDL in in one one of of these these crystals. crystals. FigureFigure 1.1.TEM TEM imageimage ofof humanhuman toothtooth enamel.enamel. TheThe InsetInset showsshows thethe high-resolutionhigh-resolution TEMTEM (HRTEM)(HRTEM) image of one of these crystals. The arrows indicate the central dark line (CDL) positions. image of one of these crystals. The arrows indicate the central dark line (CDL) positions. BecauseBecause enamelenamel crystalscrystals beginbegin toto dissolvedissolve fromfrom thethe centercenter duringduring acidicacidic attacksattacks suchsuch asas cariescaries [ 6[6],], the the CDL CDL is is of of particular particular interest. interest. Figure Figure2a 2 showsa shows the the graphic graphic representation representation of theof the dissolution dissolution of the of humanthe human tooth tooth enamel enamel by acid by attack, acid andattack Figure, and2 bFigure shows 2 itsb sho HRTEMws its imageHRTEM as theyimage began as they to dissolve began afterto dissolve treated withafter orthophosphoric treated with orthophosphoric acid for simulating acid acid for attacks:simulating the acid acid attacks attack: always the acid started attack atalways the center started of at the the crystals. center of Taking the crystals. as a basis Taking the hexagonalas a basis the unit hexagonal cell of HAP, unit during cell of caries, HAP, the during human caries tooth, the enamel human crystals tooth wereenamel destroyed crystals systematically:were destroyed first, systematically: a central lesion first, elongated a central alonglesion theelongated [11–20] along direction the appeared[11–20] direction on the basalappear (0001)ed on planes; the basal and (0001) secondly, planes; the and lesion secondly, developed the lesion anisotropically developed along anisotropically the [0001] directionalong the across [0001] the direction crystals across [7]. the crystals [7]. TheThe TEMTEM observationsobservations ofof thethe enamelenamel crystalscrystals indicatedindicated thatthat thethe CDLCDL waswas inin factfact aa planarplanar defect.defect. SeveralSeveral hypotheseshypotheses havehave beenbeen proposedproposed inin itsits possiblepossible origin.origin. OneOne ofof themthem isis thatthat thethe CDLCDL isis aa transformation-mismatchedtransformation-mismatched layerlayer fromfrom octacalciumoctacalcium phosphatephosphate (OCP)(OCP) toto HAP.HAP. However, However, the the HAP–OCP HAP–OCP interface interface model model generated generated doesdoes notnot fullyfully reproducereproduce thethe TEMTEM datadata [ 8[8].]. Another Another possibility possibility is that is that the CDLthe CDL is a Ca-rich is a Ca region-rich withinregion enamelwithin crystalsenamel andcrystals therefore and therefore cannot be cannot the residual be the ofresidual the OCP–HAP of the OCP mismatch–HAP mismatch [8,9]. [8,9]. The contrast of the CDL, when observed with a scanning transmission electron mi- croscope (STEM) using an annular detector, gave additional clues on the CDL’s structure. Modern STEM microscopes were equipped with aberration correction systems, and the low- and high-angle scattered electron annular detectors provided bright-field (BF), low-angle annular dark-field (ADF), and high-angle annular dark-field (HAADF) images with sub- Appl. Sci. 2021, 11, 7477 3 of 13 angstrom resolution [10]. STEM-BF images include the transmitted beam and small-angle scattered beams produced by Rutherford elastic scattering, plasmons, and phonons [11]. When human tooth enamel crystals were observed by an STEM, the BF image showed a contrast
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