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 tooth tooth 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 Gaussian Gaussian 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 human the human tooth tooth enamel enamel by acid by attack, acid andattack Figure, and2 b Figure shows 2 itsb sho HRTEMws its imageHRTEM as they image began as they to dissolve began after to dissolve treated with after 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 data data [ 8[8].]. Another Another possibility possibility is that is that the CDL the CDLis a Ca-rich is a Ca region-rich within region enamel within crystals enamel 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 microscope (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 similar to the HRTEM images, and the CDL appeared dark (Figure3a). In the STEM-ADF image, the collection of scattered beams was made at angles between 25 and 250 mrad and, therefore, their contrast present elastic deformation ﬁelds (Figure3b) [ 11]. In this case, the CDL showed a bright contrast surrounded by an extended gray zones produced by strain (Figure3d) [ 2,3]. For the HAADF-STEM image, scattered beams at angles between 50 and 250 mrad were collected. These large-angle scattered beams were related to the atomic number of the sample (the Z-contrast), and the contrast of these images at atomic resolution was “interpretable” in a direct way [11]. That is, the light contrast indicated atomic positions with a larger Z value, and the gray and dark contrast were related to atomic positions with a smaller Z value. Figure3c shows the HAADF-STEM image of the human tooth enamel crystal at a low magniﬁcation. In this case, the CDL Appl. Sci. 2021, 11, x FOR PEER REVIEWcontrast was weak and, sometimes, becomes almost invisible, which indicated that3 of the 14 large-angle scattered beams were generated in practically the whole crystal.

(a) (b)

FigureFigure 2. 2. ((aa)) Graphic Graphic representation representation of of the the dissolution dissolution of of the the enamel enamel crystals crystals of of human human teeth teeth by by acid acid attack. attack. Th Thisis starts starts fromfrom the the center center of of the the crystal. crystal. (b ()b HRTEM) HRTEM image image of ofenamel enamel crystals crystals in step in step 2 of 2 a ofn enamel an enamel sample sample after after treated treated with with or- thophosphoricorthophosphoric acid acid forfor simulating simulating acid acid attacks attacks..

TheThe contrast electron of diffraction the CDL,patterns when observed (EDPs) ofwith human a scanning tooth enamel transmission crystals electron indicated mi- a croscopehexagonal (STEM) unit cell using in agreementan annular withdetector the, HAPgave additional data of a PDF clues 09–0432 on the CDL X-ray’s diffractionstructure. Moderncard (hexagonal STEM microscopes unit cell with were lattice equipped parameters with aberration (a = 9.418 Åco andrrection c = 6.884 systems Å) and, and space the lowgroup- and P63/m high (No.-angle 176)). scattered Figure 4 electrona shows the annular electron detectors diffraction provide patternd bright of a human-field tooth (BF), lowenamel-angle crystal annular along dark the-field [10-10] (ADF) direction, and high (the- diffractionangle annular patterns dark- shownfield (HAADF) in Figure 4images are in withfact nano-diffractionsub-angstrom resolution patterns (nano-EDP),[10]. STEM-BF since images the crystals include had the nanometrictransmitted dimensions beam and smalland the-angle minimum scattered cross-section beams produced of the incident by Rutherford electron beamelastic used scattering, to obtain plasmons, EDPs in TEM and phononswas greater [11]. than When 10 nm human and tooththe nanometric enamel crystals region were of the observed crystals from by an which STEM, the the EDPs BF imagecame was show practicallyed a contrast the whole similar crystal to the of HRTEM the human images tooth, and enamel). the CDL The CDLappear planeed dark was (Figureperpendicular 3a). In the to theSTEM [10-10]-ADF direction image, the and collection parallel to of the scattered plane formed beams bywas the made [0001] at andan- gles[1-210] between directions 25 and (Figure 250 mrad4b). Therefore, and, therefore, for the their observation contrast of present the CDL, elastic the crystalsdeformatio mustn fieldsbe oriented (Figure in 3 directionsb) [11]. In perpendicularthis case, the CDL to the show [10-10]ed a axis, bright such contrast as the [0001],surrounded [10-11], by andan extended[1-210] directions, gray zones and produced these were by strain the directions (Figure 3 ind) which[2,3]. For the the crystals HAADF were-STEM observed image, to scatteredobtain the beams STEM at images angles that between we presented 50 and in250 this mrad work. were If the collected. [0001] axis These was large designated-angle ◦ ◦ ◦ scatteredas 0 , the beams [10-11] were and [1-210]related axes to the were atomic at 54 numberand 90 of, respectively. the sample (the Z-contrast), and the contrast of these images at atomic resolution was “interpretable” in a direct way [11]. That is, the light contrast indicated atomic positions with a larger Z value, and the gray and dark contrast were related to atomic positions with a smaller Z value. Figure 3c shows the HAADF-STEM image of the human tooth enamel crystal at a low magnification. In this case, the CDL contrast was weak and, sometimes, becomes almost invisible, which indicated that the large-angle scattered beams were generated in practically the whole crystal. Appl. Sci. 2021Appl.Appl., 11 Sci. Sci., 747720 202121, ,11 11, ,x x FOR FOR PEER PEER REVIEW REVIEW 44 of of 14 14 4 of 13

. . ((aa)) ((bb)) ((cc))

((dd))

Figure 3. Scanning transmissionFigure electronFigure 3. 3. Scanning Scanning microscopy transmission transmission (TEM) electron imageselectron microscopy microscopy of the human (TEM) (TEM) tooth images images enamel of of the the human crystals.human tooth tooth (a) enamel enamel STEM-bright- crystals.crystals. ( (aa)) STEM STEM--brightbright--fieldfield (BF) (BF) image; image; ( (bb)) STEM STEM--angularangular dark dark--fieldfield (ADF) (ADF) image; image; ( (cc)) field (BF) image; (b) STEM-angularSTEMSTEM dark-field--highhigh--angleangle (ADF) angular angular image; dark dark- (-fieldcfield) STEM-high-angle (HAADF) (HAADF) image. image. Note Note angular the the CDL CDL dark-field contrast contrast in (HAADF)in each each case; case; image. ( (dd)) Note theAppl. CDL Sci. 20 contrast21, 11, x FOR in PEER each REVIEW case; ( dmagnified)magnified magnified ADF ADF ADF-STEM--STEMSTEM image image image of of one one ofof the the one human human of the tooth tooth human enamel enamel tooth crysta crysta enamellsls where where crystals the the dark dark where contrast 5contrast of 14 the dark

contrast around the CDL indicatesaroundaround a region the the CDL CDL of strain.indicates indicates The a a region region arrows of of strain. indicatestrain. The The thearrows arrows CDL indicate indicate positions. the the CDL CDL positions. positions.

TheThe eelectronlectron diffractiondiffraction patternspatterns (EDPs)(EDPs) ofof humanhuman toothtooth enamelenamel crystalscrystals indicateindicatedd aa hexagonalhexagonal unitunit cellcell inin agreementagreement withwith thethe HAPHAP datadata ofof aa PDFPDF 0099––04320432 XX--rayray diffractiondiffraction cardcard (hexagonal(hexagonal unitunit cellcell withwith latticelattice parametersparameters ((aa == 9.4189.418 ÅÅ anandd cc == 6.8846.884 ÅÅ)) andand spacespace groupgroup P63/m P63/m (No. (No. 176)). 176)). Figure Figure 4 4aa showsshows the the electron electron diffraction diffraction pattern pattern of of a a human human toothtooth enamelenamel crystalcrystal alongalong thethe [10[10--10]10] directiondirection (the(the diffractiondiffraction patternspatterns shownshown inin FigureFigure 44 areare inin factfact nanonano--diffractiondiffraction patternspatterns (nano(nano--EDP)EDP), , sincesince thethe crystalscrystals hahadd nanometricnanometric di-di- mensionsmensions andand thethe minimumminimum crosscross--sectionsection ofof thethe incidentincident electronelectron beambeam usedused toto obtainobtain EDPsEDPs in in TEM TEM waswas greatergreater than than 10 10 nmnm and and the the nano nanometricmetric region region of of the the crystals crystals from from whichwhich thethe EDPsEDPs ccaameme waswas practicallypractically thethe wholewhole crystalcrystal ofof thethe humanhuman toothtooth enamel).enamel). TheThe CDLCDL plane plane was was perpendicular perpendicular to to the the [10 [10--10]10] direction direction and and parallel parallel to to the the plane plane formed formed by by thethe [0001][0001] andand [1[1--210]210] directionsdirections (Figure(Figure 44bb).). Therefore,Therefore, forfor thethe observationobservation ofof thethe CDL,CDL, thethe crystalscrystals mustmust bebe orientedoriented inin directionsdirections perpendicularperpendicular toto thethe [10[10--10]10] axis,axis, suchsuch asas thethe [0001],[0001], [10[10--1111],], andand [1[1--210]210] directions,directions, andand thesethese werewere thethe directionsdirections inin whichwhich thethe crystalscrystals werewere observedobserved toto obtainobtain thethe STEMSTEM imagesimages thatthat wewe presentpresenteded inin thisthis work.work. IfIf thethe [[0001]0001] axisaxis was was designated designated as as 0°, 0°, the the [10 [10--11]11] and and [1 [1--210]210] axes axes were were at at 54° 54° and and 90°, 90°, respectively. respectively.

(a) (b)

FigureFigure 4. 4. (a(a) )Electron Electron diffraction diffraction pattern pattern of a human of a human tooth enamel tooth crystal enamel along crystal the [10 along-10] axis. the It [10-10] axis. It was indexed with the hexagonal hydroxyapatite (HAP) unit cell. (b) The [1000], [10-11], and [1-210] wasdiffraction indexed patterns, with which the hexagonal are perpendicular hydroxyapatite to the [10- (HAP)10] axis. unitThe color cell. of (b the) The line [1000], indicates [10-11], the and [1-210] diffractionzone axis of the patterns, diffraction which pattern are of perpendicular the squares with to the the same [10-10] color. axis. The color of the line indicates the zone axis of the diffraction pattern of the squares with the same color.

Figure 5. (a) Three-dimensional (3D) representation of the hexagonal HAP unit cell indicating the position of the CDL plane (the [10-10] plane). (b) View along the [0001] axis. The rectangle indicates the position of the CDL. (c) View along the [1-011] axis. Ca atoms are in blue, P atoms are in green, O atoms are in red, and H atoms are in yellow.

1.3. Dental Caries and Defects at the Nanoscale Dental caries and periodontal diseases are the most common bacterial infections in humans [12]. As observed in Figure 2, at the nanoscale, during carious processes or other Appl. Sci. 2021, 11, x FOR PEER REVIEW 5 of 14

Appl. Sci. 2021, 11, 7477 5 of 13

Figure5 shows(a) a three-dimensional (3D) representation(b) of the CDL plane in the hex- Figureagonal 4. (a) HAPElectron unit diffraction cell. The pattern CDL ofplane a human coincided tooth enamel with crystal the planealong the formed [10-10] by axis. [0001] It and was[1-210], indexed i.e.,with the the planehexagonal (10-10). hydroxyapatite Figure5 also(HAP includes) unit cell. the(b) The views [1000], of the [10- HAP11], and unit [1-210] cell along diffractionthe [0001] patterns, (Figure which5b) are and perpendicular [10-11] (Figure to the5b) [10 axes.-10] axis. Asobserved The color of in the Figure line 5indicatesb, the HAP the unit zonecell axis was of the symmetric diffraction onpattern both of sides the squares of the with CDL the plane, same whichcolor. became a mirror plane m.

FigureFigure 5. (a 5.) Three(a) Three-dimensional-dimensional (3D) representation (3D) representation of the ofhexagonal the hexagonal HAP unit HAP cell unit indicating cell indicating the the positionposition of the of CDL the CDL plane plane (the [ (the10-10 [10-10]] plane plane).). (b) View (b) along View the along [0001] the axis. [0001] The axis. rectangle The rectangle indicates indicates the theposition position of the of CDL the CDL.. (c) View (c) View along along the [1 the-011] [1-011] axis. axis.Ca atoms Ca atoms are in are blue, in blue,P atoms P atoms are in aregreen, in green, O O atomsatoms are are in in red, red, and and H Hatoms atoms are are in yellow. in yellow.

1.3.1.3. Dental Dental Caries Caries and Defects and Defects at the at Nanoscale the Nanoscale DentalDental caries caries and periodontal and periodontal diseases diseases are the aremost the common most common bacterial bacterialinfections infectionsin humansin humans [12]. As [12 observed]. As observed in Figure in 2, Figureat the nanoscale,2, at the nanoscale, during carious during process cariouses or processes other or other acidic attack, human tooth enamel crystals were destroyed systematically: first a central lesion elongated along the [11-20] direction appeared on the (0001) basal planes of the crystals; secondly, this lesion developed anisotropically along the [0001] direction across the crystals; and third, the crystals were break open [7]. No full explanation for the anisotropic crystal dissolution has yet been put forward. Neither the structural reason for the anisotropic dissolution nor the implications for the mineralization/demineralization process have yet been fully understood. Nevertheless, several common-sense observations have been made (Figure2): (1) there was a structural feature on the center of the crystal basal faces different from the adjoining surface; (2) after dissolution, the lesion was hexagonal in shape and did not split the crystal along the [11-20] direction; (3) the dissolution process propagated anisotropically along the [0001] direction, which indicated that the structural feature was 3D. Several approaches are followed to conduct the ongoing research on that subject such as the analysis of etch pits at the HAP surface [13,14] and surface energy calculations [15]. Defects in tooth enamel crystals aligned along the [11-20] direction have been analyzed by HRTEM. Brès et al. [6] identified some types of disorders that may act as dissolution sites. Recent work by Gordon et al. [16] and by Yun et al. [3] have shown a high concentration of Na+ and Mg2+ ions at the center of enamel crystals. These findings have been specified by DeRocher et al. [2], who have shown the existence of two nanometric layers enriched 2+ + – 2− with Mg flanking a core rich in Na ,F , and CO3 ions. This sandwich core being Appl. Sci. 2021, 11, 7477 6 of 13

surrounded by a shell with lower concentrations of substitutional defects. The residual stress created by the chemical gradients may favor the anisotropic carious dissolution. All these works are still in progress. It must be noted that no explanation has yet been given on the elongated shape of the lesion along the [11-20] direction and for the fact that it does not split open the crystals as would a grain boundary. Another approach to understanding the structural mechanisms of the dissolution anisotropy is the analysis of a contrast phenomenon observed at the very dissolution sites such as the CDL. In this paper, we present the analysis of the aberration-corrected high-resolution HAADF-STEM images of the CDL of human tooth enamel crystals along the [0001] (0◦/a), [10-11] (54◦/a), and [1-210] (90◦/a) directions, which will allow obtaining more detailed information on the structural characteristics of the CDL at the nanometric level.

2. Materials and Methods Human tooth samples of 30-year-old permanent molar teeth obtained from dental treatments were used according to the Institutional Review Board (IRB) approval protocol (FMED/CI/SPR/083/2015) approved by the University of Mexico. The general sample preparation procedure consisted of the following: (1) cutting the tooth, (2) mechanical thin- ning, (3) polishing to mirror finish, (4) etching with orthophosphoric acid, and (5) cutting with a focused beam (FIB). A careful selection of teeth was made to avoid those with cracks and/or severe decay damage. Human tooth enamel was cut with a diamond disc into 3 × 3 mm slices with 250 to 500 µm thickness. The thickness reduction was carried out by mechanical polish down with a sandpaper to thicknesses less than 100 µm. The samples were mirror-polished with a micro cloth and alumina powder. To clean the sample, it was placed in a solution of isopropanol and acetone (v/v: 60%:40%) in the ultrasonic cleaner for 15 min. The mirror- finished sample was etched with phosphoric acid to reveal its structure. Finally, the sample was washed with distilled water for 10 min and dried with compressed air. With the FIB equipment of gallium (Ga) ions, foils of the sample with an adequate TEM thickness were obtained. At the end, the foil was set on a TEM grid for observation. A Thermo Fisher Scientific QUANTA 200-3D FIB (Thermo Fisher Scientific, Hillsboro, OR, USA) with a field emission filament (FIB) and a Ga ion source was used. The acceleration voltage was 30 kV for the two beams at probe currents of 200 nA for the electron beam and 50 nA for the ion beam. The electron and Ga ion beams coincided at a working distance of 10 mm. The OmniprobeTM (Oxford Instruments, Abingdon, UK) 100.7 micromanipulator equipped with gas injection systems for platinum (Pt) deposition was used for the in-situ manipulation of TEM samples. STEM images were obtained with the FEI Qu-Ant-EM microscope with Cs correction at the EMAT of the University of Antwerp. This was an FEI Titan G3 microscope (FEI, Eindhoven, The Netherlands) equipped with a Schottky field emission electron gun and spherical aberration correctors. The double aberration correctors offered a spatial resolution of 0.5 Å with TEM and that of 0.8 Å with STEM and a flexible choice of acceleration voltages between 60 and 300 kV. For enamel samples observations, we worked with 200 kV and with the lowest electron dose, <0.1 e/A2, because the electron damage in enamel samples was greater at 60 kV and 80 kV than at 200 kV (result commonly observed in ceramic materials in which radiolysis damage and heating are registered [17]). The main observation parameters were as following: voltage, 200 kV; spot size, 9; probe current, 0.04 nA; and working length, 56–91 mm for HAADF observations with internal detection angles of 41 to 92 mrad and 220–230 mm for HAADF observations with internal detection angles of 10 to 35 mrad for ADF observations. Because low-dose STEM images are very noisy, the image contrast was increased by digital processing using the Fast Fourier Transform (FFT) and dot filters with the GATAN’s Digital-Micrograph software (Gatan Inc., Boston, MA, USA). JEMS software [18] was used for the simulation. Appl. Sci. 2021, 11, 7477 7 of 13

3. Results Considering that the STEM-HAADF images depend on the atomic number of the sample and that Z = 20 for Ca, Z = 15 for P, Z = 8 for O, and Z = 1 for H, the bright spots are related to the Ca atoms, followed in intensity by the P atoms. The O and H atoms had the minimum intensity. This made the contrast of these images to be directly interpreted. Figure6a shows the STEM-HAADF image of human tooth enamel crystals in the [0001] direction. The crystalline periodicity can be clearly observed in the enamel crystals, and neither planar defects nor dislocations were directly observed in the CDL region. Taking that the brightest points corresponded to the Ca atom positions in the HAP unit Appl. Sci. 2021, 11, x FOR PEER REVIEW 8 of 14 cell, and the line indicates the CDL position, Figure6b shows the arrangement generated (the voids presented were deliberately introduced for the clarity of the arrangement). The periodicities observed along the H, LI, and LR lines (Figure6c), which were parallel in the crystalshexagon, and no sides, planar were defects 8.02 ±nor0.66 dislocations Å, 8.26 ± were0.26 observed Å, and 8.19 in the± 0.27CDL Å, region. respectively, The while for arrangementHAP free obtained of defects, with this the periodicity HAP unit cell was by 8.17 the Å.superposition These periodicities of the bright implied, spots therefore, the withexistence the Ca atoms of stress is presented or strain in Figure ﬁelds in8b. the Figure tooth 8c enamelshows the crystal. periodicities The superposition observed in of the HAP Figure 8a along the V and H directions, which were parallel on the rectangle sides. These atom positions is another fact observed. For a better observation of the mentioned overlap, were 8.17 ± 0.72 Å and 3.44 ± 0.25 Å, respectively. Figure 8d shows again an enlargement of FigureFigure 8b6 aroundd shows the an CDL, enlargement and the overlapping of Figure 6observedb around was the produced region through by the strain which the CDL fieldswent. present The in overlappingthe atomic arrangement produced isof athe direct crystals. result The of thickness the strain of ﬁeldsthe CDL present in this in the atomic case arrangementwas 4.7 Å. of the crystals. The thickness of the CDL in the STEM image was 6.17 Å.

FigureFigure 6. (a) 6.STEM(a)- STEM-HAADFHAADF image of image human of tooth human enamel tooth crystal enamels in the crystals [0001] indirection. the [0001] (b) Ar- direction. (b) Ar- rangement obtained by identifying the bright points with the Ca atoms of HAP. The voids are presentedrangement just for obtainedthe clarity byof the identifying arrangement. the Note bright the pointssuperposition with theof the Ca atoms atoms positions. of HAP. (c)The voids are Periodicitiespresented observed just for along the the clarity H, LI, of and the LR arrangement. lines separately Note. (d) Magnification the superposition of the CDL of the zone; atoms positions. the observed(c) Periodicities thickness observed of the CDL along is also the indicated. H, LI, and The LR blue lines circle separately. is at the ( dsame) Magniﬁcation position in the of the CDL zone; threethe cases. observed The yellow thickness line indicates of the the CDL position is also of indicated.the CDL. Ca The atoms blue are circle in blue, is atP atoms the same are in position in the green, O atoms are in red, and H atoms are in yellow. three cases. The yellow line indicates the position of the CDL. Ca atoms are in blue, P atoms are in green, O atoms are in red, and H atoms are in yellow.

Figure7a shows the STEM-HAADF image of human tooth enamel crystals in the [10-11] direction. Again, the crystalline periodicity was observed in the enamel crystals, and neither planar defects nor dislocations were observed in the CDL region. Considering the brightest points corresponding to the Ca positions in the HAP unit cell, the arrangement shown was generated. As for the [0001] direction, note the superposition that occurred on the positions of the cells, as shown in Figure7b. Figure8c shows the periodicities observed in Figure7a along the V and H lines, which were parallel to the rectangle sides, which were Appl. Sci. 2021, 11, 7477 8 of 13

8.17 ± 0.51 Å and 5.54 ± 0.41 Å, respectively. Figure7d shows again an enlargement of Figure7B around the CDL. The overlapping was produced by the strain ﬁelds present in Appl.Appl. Sci. Sci. 20 2120,21 11, ,11 x ,FOR x FOR PEER PEER REVIEW REVIEW 9 of9 14of 14 the atomic arrangement of the crystals. The thickness of the CDL observed in the STEM image was 3.7 Å.

FigureFigureFigure 7. (7.a ) 7.( aSTEM)( aSTEM) STEM-HAADF-HAADF-HAADF image image of image ofhuman human of tooth human tooth enamel enamel tooth crystal enamelcrystals ins crystalsinthe the [10 [10-11] in-11] direction. the direction. [10-11] (b )( direction.bAr-) Ar- (b) Ar- rangementrangementrangement obtained obtained obtained by by identifying identifying by identifying the the bright bright the points brightpoints with pointswith the the Ca with Ca atoms atoms the in Ca inthe atomsthe HAP HAP inunit unit the cell. HAPcell. The Theunit cell. The voids are presented for the clarity of the arrangement. (c) The periodicities observed along the V voidsvoids are are presented presented for for the the clarity clarity of the of thearrangement. arrangement. (c) The (c) periodicities The periodicities observed observed along the along V the V and andand H Hdirections directions separately separately. (d. )( dMagnifi) Magnified edCDL CDL zone; zone; the t heobserved observed thickness thickness of ofthe the CDL CDL is indi-is indi- H directions separately. (d) Magniﬁed CDL zone; the observed thickness of the CDL is indicated. cated.cated. The The blue blue rectangle rectangle is atis atthe the same same position position in inthe the three three cases. cases. The The yellow yellow line line indicates indicates the the positionpositionThe blueof ofthe the rectangle CDL. CDL. Ca Ca isatoms atatoms the are sameare in inblue, positionblue, P atomsP atoms in are the are in three ingreen, green, cases. O atomsO Theatoms yelloware are in inred, line red, and indicates and H Hatoms atoms the position of areare inthe inyellow. CDL.yellow. Ca atoms are in blue, P atoms are in green, O atoms are in red, and H atoms are in yellow.

FigureFigureFigure 8. (8.a ) 8.( aSTEM)( aSTEM) STEM-HAADF-HAADF-HAADF image image of image ofhuman human of tooth human tooth enamel enamel tooth crystal enamelcrystals ins crystalsinthe the [1 -[1210]-210] in direction. the direction. [1-210] (b )( direction.bAr-) Ar- (b) Ar- rangement obtained by identifying the bright points with the Ca atoms in the HAP unit cell. (c) The rangementrangement obtained obtained by identifying by identifying the bright the bright points pointswith the with Ca atoms the Ca in atoms the HAP in unit the HAPcell. (c unit) The cell. (c) The periodicitiesperiodicities along along the the V Vand and H Hdirections directions separately separately. (d. ) ( d Magnifi) Magnifieded CDL CDL zone. zone. The The observed observed periodicities along the V and H directions separately. (d) Magniﬁed CDL zone. The observed thick- thicknessthickness of ofthe the CDL CDL is indicated.is indicated. The The blue blue rectangle rectangle is atis atthe the same same position position in inthe the three three cases. cases. Th The e yellowyellowness line ofline indicates the indicates CDL the is the indicated.position position of ofthe The the CDL. blue CDL. Ca rectangle Ca atoms atoms are is are in at inblue, the blue, same P atoms P atoms position are are in ingreen, green, the O three aOtoms atoms cases. in in The yellow red,red,line and and indicatesH atomsH atoms are the are in positioninyellow. yellow. of the CDL. Ca atoms are in blue, P atoms are in green, O atoms in red, and H atoms are in yellow. Appl. Sci. 2021, 11, 7477 9 of 13

Figure8a shows the STEM-HAADF image of human tooth enamel crystals in the [1-210] direction. Once again, the crystalline periodicity was observed in the enamel crystals, and no planar defects nor dislocations were observed in the CDL region. The arrangement obtained with the HAP unit cell by the superposition of the bright spots with the Ca atoms is presented in Figure8b. Figure8c shows the periodicities observed in Figure8a along the V and H directions, which were parallel on the rectangle sides. These were 8.17 ± 0.72 Å and 3.44 ± 0.25 Å, respectively. Figure8d shows again an enlargement of Figure8b around the CDL, and the overlapping observed was produced by the strain ﬁelds present in the atomic arrangement of the crystals. The thickness of the CDL in this case was 4.7 Å.

4. Discussion 4.1. TEM and STEM Contrasts The high-resolution STEM-HAADF images of human tooth enamel crystals presented in this work indicated that the CDL thickness was between 3 and 8 Å. The CDL plane was formed by the [1-210] and [0001] directions, and it divided the enamel crystal into two parts, which were related by a mirror plane m. Table1 summarizes the contrasts presented by the CDL when it was observed by TEM, HRTEM, and STEM in BF and dark-field (DF) modes.

Table 1. CDL contrast observed when it is viewed by TEM, HRTEM, and STEM in the BF and dark ﬁeld (DF) modes.

Image TEM HTEM STEM Amplitude Phase Amplitude Contrast (1-beam) (N-beams) (N-beams) Mode BF DF BF BF ADF HAADF Contrast Dark or white Dark Dark Dark White color (defocusing function) (but weak)

In TEM, the CDL behaved as a Fresnel contrast, because it changed from dark in overfocus conditions to bright in underfocus conditions, and disappears at the Gaussian focus. This Fresnel contrast suggests a change in the internal mean potential produced by the local deviation in stoichiometry in the central region of the enamel crystal [6] similar to the one presented in the TEM by the oriented-in-edge-on-direction grain boundary dislocations and voids [19]. Thus, the origin of the CDL would be a local deviation in stoichiometry in the HAP unit cell at the center of the enamel crystal. The STEM-ADF contrasts suggested that enamel crystals had a “core-shell” type structure (Figure3d). DeRocher et al. [ 2] indicated that the dark contrast around the CDL was a strained zone and the light contrast in the periphery was free of strain. DeRocher et al. [2] and Yun et al. [3] have also indicated that the dark zone is rich in Mg and Na. The presence of Mg2+ in human tooth enamel crystals has been well established [3,20], together with 2− high carbon concentrations due to the presence of carbonate (CO3 ) ions [2,3]. Therefore, the size relationship between Ca2+ and Mg2+ results in the lattice contraction deformation observed [2]. In this way, residual stresses due to chemical gradients agree quite well with the preferential dissolution from the CDL of the enamel crystal in acidic media. Yun et al. [3] commented that an antiphase boundary (APB) can also be produced when enamel crystals grow during amelogenesis, as it happens in the monoclinic crystals [3]. The APB has higher energy than the rest of the crystal provided surely by ions such as Mg2+ and Na+, and it would also explain why CDL is susceptible to demineralization. Although the APB shows the same structure on both sides, the arrangement should be staggered by half an atomic plane. However, this displacement was not observed in the high-resolution STEM-HAADF images. Therefore, it is reasonable to believe that the contrast behavior of the CDL is an indication that in the CDL the mean internal potential is lower than in the perfect crystal and produced by the local deviation of the stoichiometry of the HAP unit cell. Appl. Sci. 2021, 11, 7477 10 of 13

4.2. Importance of Crystal Defects for the Properties of Enamel Crystals The knowledge of the ﬁne structural details favoring the dissolution anisotropy of enamel crystals during the carious process is of prime importance for the development of the medical treatment of the disease. It is striking to note that the initial dissolution

Appl. Sci. 2021, 11, x FOR PEER REVIEWtakes place at the very CDL site. Hence, any development in the knowledge on11 of the 14 CDL advances the knowledge of the dissolution anisotropy and vice versa. The existence of screw dislocations inside ionic crystals not only favors dissolution, by also growthThe existe as firstnce shown of screw by Frankdislocations [21], who inside has ionic shown crystals that dislocationsnot only favors favor dissolution low saturation, growth.by also The growth growth as first mechanism shown by isFrank favored [21], bywho the has difficulty shown that encountered dislocations by favor the crystallow to balancesaturation electrical growth. charges The atgrowth the surface mechanism [22]. Hence,is favored the by importance the difficulty is for encountered mineralization. by theFurthermore, crystal to balance in ionic electrical crystals charges such asat the HAP, surface there [22]. exists Hence a relationship, the importance between is for crystal defectsmineralization. and electric charges, as jogs on surfaces and dislocations act as sources and sinks for pointFurthermore, defects. The in establishment ionic crystals of such the as electrical HAP, there equilibrium exists a with relationship the vacancies, between solute ions,crystal and defects interstitials and electric produces charges a net, as chargejogs on surfaces on the surface and dislocations or dislocation, act as sources surrounded and by a compensatingsinks for point spacedefects. charge The establishment region. Within of the this electrical space, equilibrium the charge with region the vacancies, typically has a thicknesssolute ions, of the and order interstitials of 10 nm. produces There are a net very charge large on electric the surface ﬁelds or(up dislocation, to about 105 sur- V/cm) androunded very large by aperturbations compensating of space point charge defect region. concentrations Within this (by space factors, the of charge several region hundred). typically has a thickness of the order of 10 nm. There are very large electric fields (up to It is likely that such electric ﬁelds are related to the change in mean inner potential that about 105 V/cm) and very large perturbations of point defect concentrations (by factors of giveseveral rise to hundred). the CDL It [23 is, 24likely]. that such electric fields are related to the change in mean innerIt is potential also likely that that give the rise surface to the chargeCDL [23,24]. at the emergence of dislocations on ionic surfaces can favorIt is the also adhesion likely that of the proteins surface [ 25charge]. at the emergence of dislocations on ionic surfaces can favor the adhesion of proteins [25]. 4.3. Electron Radiation Damage 4.3.It Electron is also well-knownRadiation Damage that human tooth enamel crystals are susceptible to electron beam radiationIt is also damagewell-known during that human TEM observation tooth enamel [13 crystals,17]. As are an susceptible example ofto this,electron Figure 9 showsbeam the radiation high-resolution damage during STEM-HAADF TEM observation image [13,17]. of the humanAs an example tooth enamel of this, crystalsFigure 9 along theshows [0001] the direction high-resolution with higher STEM electron-HAADF damage. image of The the generated human tooth arrangement enamel crystal obtaineds by identifyingalong the [0001] the direction bright points with higher with theelectron Ca atoms damage. of theThe HAP generated unit cellarrangement showed ob- a higher densitytained of by defects identifying and the a major bright overlappingpoints with the of Ca the atoms atoms. of the Therefore, HAP unit cell the show TEMed electron a beamhigher increased density the of defects overlap and of athe major HAP overlapping atoms during of the the atoms. observation Therefore, of the human TEM tooth electron beam increased the overlap of the HAP atoms during the observation of human enamel crystals. On the other hand, Figure 10 shows the STEM-HAADF image of human tooth enamel crystals. On the other hand, Figure 10 shows the STEM-HAADF image of toothhuman enamel tooth crystals enamel crystal alongs the along [0001] the [0001] direction direction but but with with drastic drastic electron electron damagedamage and, therefore,and, therefore, higher defect higher density. defect density. Therefore, Therefore, to have to a have better a better interpretation interpretation of the of structural the characteristicsstructural characteristics of the CDL of and the CDL the humanand the human tooth enamel tooth enamel crystal crystal structure, structure, a way a way should be soughtshould be to sought reduce to or reduce avoid orthis avoid situation. this situation. One One possible possible way way to to reduce reduce this this effecteffect is by coatingis by coating the sample the sample surface surface with awith layer a layer of amorphous of amorphous carbon. carbon.

(a) (b)

FigureFigure 9. (a) STEM-HAADF9. (a) STEM-HAADF image image of humanof human tooth tooth enamel enamel crystalscrystals in in the the [0001] [0001] direction direction with with electron electron damage. damage. (b) Ar- (b) Ar- rangement obtained by identifying the bright points with the Ca atoms of the HAP unit cell. The voids presented were rangement obtained by identifying the bright points with the Ca atoms of the HAP unit cell. The voids presented were generated for clarity. The blue circle is at the same position in both cases. The yellow line indicates the position of the generatedCDL. for Ca clarity. atoms are The in blue blue, circle P atoms isat are the in samegreen,position O atoms inare both in red, cases. and H The atoms yellow are in line yellow. indicates the position of the CDL. Ca atoms are in blue, P atoms are in green, O atoms are in red, and H atoms are in yellow. Appl. Sci. 2021, 11, 7477 11 of 13 Appl. Sci. 2021, 11, x FOR PEER REVIEW 12 of 14

Figure 10. STEM-HAADF image of human tooth enamel crystals along the [0001] direction with Figure 10. STEM-HAADF image of human tooth enamel crystals along the [0001] direction with severe electron beam damage. severe electron beam damage.

ItIt isis importantimportant toto mentionmention herehere thatthat inin thethe resultsresults presentedpresented inin thisthis work,work, aa simplesimple superpositionsuperposition of of images images of of the the hydroxyapatite hydroxyapatite unit unit cell cell was was performed performed in thein the mentioned mentioned directiondirection and and we havewe have not performed not performed an atomic an atomic simulation. simulation. In addition, In addition, although although the overlap the producedoverlap produced is the direct is the result direct of the result deformation of the deformation ﬁelds present fields in the present atomic in arrangement the atomic ar- of therangement human toothof the enamel human crystals, tooth enamel we must crystals, ﬁnd a we way must to perform find a way an atomicto perform simulation an atomic in thesimulation near future in the because near future it is necessary. because it is necessary.

5.5. ConclusionsConclusions Aberration-correctedAberration-corrected high-resolutionhigh-resolution HAADF-STEMHAADF-STEM imagesimages indicateindicate thatthat thethe CDLCDL thicknessthickness isis between 3 3 and and 8 8 Å, Å, depending depending on on the the axis axis of ofobservation. observation. The The CDL CDL plane plane was wasthe plane the plane formed formed by the by [0001] the [0001] and and[1-210] [1-210] directions, directions, i.e., the i.e., plane the plane (10-10). (10-10). This plane This planedivide dividedd the enamel the enamel crystal crystal into two into parts two parts,, which which were were related related by a bymirror a mirror plane plane m, im-m, implyingplying a reduction a reduction in in the the symmetry symmetry of of the the HAP HAP unit unit cells cells on on which which the the CDL CDL was was located. located. TheThe darkdark contrastcontrast observedobserved inin thethe ADF-STEMADF-STEM imagesimages andand thethe superpositionsuperposition ofof thethe atomsatoms ofof thethe HAPHAP unitunit cellcell impliedimplied thethe existenceexistence ofof stressstress andand strainstrain aroundaround thethe CDL,CDL, suggestingsuggesting aa “core-shell”-type“core-shell”-type structurestructure forfor enamelenamel crystals.crystals. TheThe FresnelFresnel contrastcontrast ofof thethe CDLCDL inin TEMTEM images,images, inin whichwhich itit changedchanged fromfrom darkdark inin thethe overfocusoverfocus andand brightbright inin thethe underfocusunderfocus conditionsconditions andand disappeareddisappeared atat thethe GaussianGaussian focus,focus, suggestedsuggested aa changechange inin thethe meanmean internalinternal potentialpotential ofof thethe crystalcrystal atat thisthis sitesite producedproduced byby thethe locallocal deviationdeviation ofof thethe stoichiometrystoichiometry ofof thethe HAPHAP unitunit cell.cell. CareCare shouldshould bebe takentaken toto reducereduce electronelectron radiationradiation damagedamage inin toothtooth enamelenamel samplessamples ratherrather thanthan introduceintroduce additionaladditional parametersparameters duringduring thethe studystudy ofof thethe structurestructure ofof thethe CDLCDL andand ofof thethe crystalcrystal ofof humanhuman toothtooth enamelenamel itself.itself. WeWe proposed proposed that that the the ﬁndings findings described described inthe in presentthe present paper paper provide provide further further informa- in- tionformation on the on structure the structure details details at the at center the center of enamel of enamel crystals; crystals; these these details details favor favor both both the anisotropicthe anisotropic carious carious dissolution dissolution and the and CDL. the However,CDL. However, we are we very are conscious very conscious that further that workfurther is requiredwork is required before a before full understanding a full understanding of both phenomena of both phenomena can be reached. can be reached.

Author Contributions: J.R.-G. and E.F.B. have done the data curation, the formal analysis, and the Author Contributions: J.R.-G. and E.F.B. have done the data curation, the formal analysis, and the writing of the manuscript. All authors have read and agreed the published version of the manuscript. writing of the manuscript. Both authors have read and agreed the published version of the manu- Funding:script. This work was ﬁnancially supported by the DGAPA-UNAM through the project PAPIIT IN101319.Funding: This work was financially supported by the DGAPA-UNAM through the project PAPIIT InstitutionalIN101319. Review Board Statement: The study was conducted according to the guidelines of theInstitutional Declaration Review of Helsinki, Board and Statement: human tooth The study samples was of conducted 30-year-old according permanent to molarthe guidelines teeth were of obtainedthe Declaration from dental of Helsinki, treatments and approved human tooth by the samples Institutional of 30- Reviewyear-old Board permanent (IRB) approval molar teeth protocol were (FMED/CI/SPR/083/2015)obtained from dental treatments approved approved by the by University the Institutional of Mexico. Review Board (IRB) approval protocol (FMED/CI/SPR/083/2015) approved by the University of Mexico. Appl. Sci. 2021, 11, 7477 12 of 13

Informed Consent Statement: It is included in the IRB approval protocol. Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable. Acknowledgments: The authors thank Gustaaf Van Tendeloo for ESTEEM support at EMAT, Uni- versity of Antwerpen (Belgium), and Johan Verbeeck for comments and suggestions. The authors also thank Stuart Torner for technical support and practical suggestions. This research received funding from the European Union Seventh Framework Programme under Grant Agreement 312483- ESTEEM2 (Integrated Infrastructure Initiative-I3). The authors also thank S. Tehuacanero-Nuñez, and P. López-Arriaga for their technical support in the elaboration of this work. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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