Abstract
Dental enamel is a principal component of teeth1, and has evolved to bear large chewing forces, resist mechanical fatigue and withstand wear over decades2. Functional impairment and loss of dental enamel, caused by developmental defects or tooth decay (caries), affect health and quality of life, with associated costs to society3. Although the past decade has seen progress in our understanding of enamel formation (amelogenesis) and the functional properties of mature enamel, attempts to repair lesions in this material or to synthesize it in vitro have had limited success4,5,6. This is partly due to the highly hierarchical structure of enamel and additional complexities arising from chemical gradients7,8,9. Here we show, using atomic-scale quantitative imaging and correlative spectroscopies, that the nanoscale crystallites of hydroxylapatite (Ca5(PO4)3(OH)), which are the fundamental building blocks of enamel, comprise two nanometric layers enriched in magnesium flanking a core rich in sodium, fluoride and carbonate ions; this sandwich core is surrounded by a shell with lower concentration of substitutional defects. A mechanical model based on density functional theory calculations and X-ray diffraction data predicts that residual stresses arise because of the chemical gradients, in agreement with preferential dissolution of the crystallite core in acidic media. Furthermore, stresses may affect the mechanical resilience of enamel. The two additional layers of hierarchy suggest a possible new model for biological control over crystal growth during amelogenesis, and hint at implications for the preservation of biomarkers during tooth development.
This is a preview of subscription content, access via your institution
Relevant articles
Open Access articles citing this article.
-
Magnesium stable isotope composition, but not concentration, responds to obesity and early insulin-resistant conditions in minipig
Scientific Reports Open Access 29 June 2022
-
Restoration of enamel anti-wear properties via remineralization: Role of occlusal loading
Friction Open Access 27 January 2022
-
Advances in biomineralization-inspired materials for hard tissue repair
International Journal of Oral Science Open Access 07 December 2021
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Rent or buy this article
Get just this article for as long as you need it
$39.95
Prices may be subject to local taxes which are calculated during checkout





Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.
Code availability
This manuscript primarily made use of commercial (IVAS, Origin, Matlab, MDI Jade, APEX2, Thermo Scientific Qtegra ISDS, COMSOL Multiphysics, TEM Imaging and Analysis, DigitalMicrograph, AZtec, Adobe Illustrator) and freely available (DEMETER, OLEX2, SHELX, Quantum ESPRESSO, Cornell Spectrum Imager, ImageJ) software packages for acquisition, processing and visualization of data. MCR was performed using custom code using the Matlab mcr.m package from the Eigenvector Research PLS_toolbox, as described elsewhere43. In addition, custom code written for the Mathematica and Matlab environments was used for file conversions, plotting and visualization. This code is available from the corresponding author upon reasonable request.
Change history
21 July 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-2544-x
References
Nanci, A. Ten Cate’s Oral Histology: Development, Structure, and Function 7th edn (Mosby Elsevier, 2008).
Chai, H., Lee, J. J. W., Constantino, P. J., Lucas, P. W. & Lawn, B. R. Remarkable resilience of teeth. Proc. Natl Acad. Sci. USA 106, 7289–7293 (2009).
Klein, O. D. et al. Meeting report: a hard look at the state of enamel research. Int. J. Oral Sci. 9, e3 (2017).
Moradian-Oldak, J. Protein-mediated enamel mineralization. Front. Biosci. 17, 1996–2023 (2012).
Lacruz, R. S., Habelitz, S., Wright, J. T. & Paine, M. L. Dental enamel formation and implications for oral health and disease. Physiol. Rev. 97, 939–993 (2017).
Robinson, C. Enamel maturation: a brief background with implications for some enamel dysplasias. Front. Physiol. 5, 388 (2014).
Gordon, L. M. et al. Amorphous intergranular phases control the properties of rodent tooth enamel. Science 347, 746–750 (2015).
La Fontaine, A. et al. Atomic-scale compositional mapping reveals Mg-rich amorphous calcium phosphate in human dental enamel. Sci. Adv. 2, e1601145 (2016).
Gordon, L. M. & Joester, D. Mapping residual organics and carbonate at grain boundaries and in the amorphous interphase in mouse incisor enamel. Front. Physiol. 6, 57 (2015).
Zhang, Y.-R., Du, W., Zhou, X.-D. & Yu, H.-Y. Review of research on the mechanical properties of the human tooth. Int. J. Oral Sci. 6, 61–69 (2014).
Robinson, C. et al. The chemistry of enamel caries. Crit. Rev. Oral Biol. Med. 11, 481–495 (2000).
Yanagisawa, T. & Miake, Y. High-resolution electron microscopy of enamel-crystal demineralization and remineralization in carious lesions. J. Electron Microsc. 52, 605–613 (2003).
Reyes-Gasga, J., Hemmerle, J. & Bres, E. F. Aberration-corrected transmission electron microscopy study of the central dark line defect in human tooth enamel crystals. Microsc. Microanal. 22, 1047–1055 (2016).
Hart, J. L. et al. Direct detection electron energy-loss spectroscopy: a method to push the limits of resolution and sensitivity. Sci. Rep. 7, 8243 (2017).
de Juan, A. & Tauler, R. Multivariate curve resolution (MCR) from 2000: progress in concepts and applications. Crit. Rev. Anal. Chem. 36, 163–176 (2006).
Gordon, L. M., Tran, L. & Joester, D. Atom probe tomography of apatites and bone-type mineralized tissues. ACS Nano 6, 10667–10675 (2012).
Gault, B., Moody, M. P., Cairney, J. M. & Ringer, S. P. Atom Probe Microscopy (Springer, 2012).
Robinson, C., Kirkham, J., Brookes, S. J., Bonass, W. A. & Shore, R. C. Int. J. Dev. Biol. The chemistry of enamel development. 39, 145–152 (1995).
Robinson, C., Weatherell, J. A. & Hallsworth, A. S. Distribution of magnesium in mature human enamel. Caries Res. 15, 70–77 (1981).
Laurencin, D. et al. Magnesium incorporation into hydroxyapatite. Biomaterials 32, 1826–1837 (2011).
Shannon, R. D. & Prewitt, C. T. Effective ionic radii in oxides and fluorides. Acta Crystallogr. B 25, 925–946 (1969).
Hughes, J. M., Cameron, M. & Crowley, K. D. Structural variations in natural F, OH, and Cl apatites. Am. Mineral. 74, 870–876 (1989).
LeGeros, R. Z., Sakae, T., Bautista, C., Retino, M. & LeGeros, J. P. Magnesium and carbonate in enamel and synthetic apatites. Adv. Dent. Res. 10, 225–231 (1996).
Ben Abdelkader, S., Khattech, I., Rey, C. & Jemal, M. Synthése, caractérisation et thermochimie d’apatites calco-magnésiennes hydroxylées et fluorées. Thermochim. Acta 376, 25–36 (2001).
Deymier, A. C. et al. Protein-free formation of bone-like apatite: new insights into the key role of carbonation. Biomaterials 127, 75–88 (2017).
Voegel, J. C. & Frank, R. M. Stages in dissolution of human enamel crystals in dental caries. Calcif. Tissue Res. 24, 19–27 (1977).
Tohda, H., Takuma, S. & Tanaka, N. Intercrystalline structure of enamel crystals affected by caries. J. Dent. Res. 66, 1647–1653 (1987).
Gao, H. J., Ji, B. H., Jager, I. L., Arzt, E. & Fratzl, P. Materials become insensitive to flaws at nanoscale: lessons from nature. Proc. Natl Acad. Sci. USA 100, 5597–5600 (2003).
Yahyazadehfar, M. et al. On the mechanics of fatigue and fracture in teeth. Appl. Mech. Rev. 66, 030803 (2014).
Yilmaz, E. D., Schneider, G. A. & Swain, M. V. Influence of structural hierarchy on the fracture behaviour of tooth enamel. Phil. Trans. R. Soc. A 373, 1–20 (2015).
Langelier, B., Wang, X. & Grandfield, K. Atomic scale chemical tomography of human bone. Sci. Rep. 7, 39958 (2017).
Stoffers, A. et. al. Correlating atom probe tomography with atomic-resolved scanning transmission electron microscopy: example of segregation at silicon grain boundaries. Microsc. Microanal. 23, 291–299 (2017).
Daculsi, G. & Kerebel, B. High-resolution electron-microscope study of human enamel crystallites — size, shape, and growth. J. Ultrastruct. Res. 65, 163–172 (1978).
Beniash, E., Metzler, R. A., Lam, R. S. & Gilbert, P. U. Transient amorphous calcium phosphate in forming enamel. J. Struct. Biol. 166, 133–143 (2009).
Robinson, C., Fuchs, P. & Weatherell, J. A. The appearance of developing rat incisor enamel using a freeze fracturing technique. J. Cryst. Growth 53, 160–165 (1981).
Kirkham, J. et al. Self-assembling peptide scaffolds promote enamel remineralization. J. Dent. Res. 86, 426–430 (2007).
Luder, H. U., Gerth-Kahlert, C., Ostertag-Benzinger, S. & Schorderet, D. F. Dental phenotype in Jalili syndrome due to a c.1312 dupC homozygous mutation in the CNNM4 gene. PLoS ONE 8, e78529 (2013).
Yamazaki, D. et al. Basolateral Mg2+ extrusion via CNNM4 mediates transcellular Mg2+ transport across epithelia: a mouse model. PLoS Genet. 9, e1003983 (2013).
Nakano, Y. et al. A critical role of TRPM7 as an ion channel protein in mediating the mineralization of the craniofacial hard tissues. Front. Physiol. 7, 258 (2016).
Aoba, T., Shimoda, S. & Moreno, E. C. Labile or surface pools of magnesium, sodium, and potassium in developing porcine enamel mineral. J. Dent. Res. 71, 1826–1831 (1992).
Hubbard, M. J., Mangum, J. E., Perez, V. A., Nervo, G. J. & Hall, R. K. Molar hypomineralisation: a call to arms for enamel researchers. Front. Physiol. 8, 546 (2017).
Park, Y. C. et al. Use of permanent marker to deposit a protection layer against FIB damage in TEM specimen preparation. J. Microsc. 255, 180–187 (2014).
Zachman, M. J., Tu, Z. Y., Choudhury, S., Archer, L. A. & Kourkoutis, L. F. Cryo-STEM mapping of solid-liquid interfaces and dendrites in lithium-metal batteries. Nature 560, 345–349 (2018).
Ravel, B. & Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 12, 537–541 (2005).
Antao, S. M., Mulder, W. H., Hassan, I., Crichton, W. A. & Parise, J. B. Cation disorder in dolomite, CaMg(CO3)2, and its influence on the aragonite + magnesite ↔ dolomite reaction boundary. Am. Mineral. 89, 1142–1147 (2004).
Dollase, W. A. & Reeder, R. J. Crystal-structure refinement of huntite, CaMg3(CO3)4, with X-ray powder data. Am. Mineral. 71, 163–166 (1986).
Calvo, C. & Gopal, R. Crystal structure of whitlockite from Palermo Quarry. Am. Mineral. 60, 120–133 (1975).
Rehr, J. J. & Albers, R. C. Theoretical approaches to X-ray absorption fine structure. Rev. Mod. Phys. 72, 621–654 (2000).
Reeder, R. J., Lamble, G. M. & Northrup, P. A. XAFS study of the coordination and local relaxation around Co2+, Zn2+, Pb2+, and Ba2+ trace elements. Am. Mineral. 84, 1049–1060 (1999).
Holt, C. et al. Preparation of amorphous calcium-magnesium phosphates at pH 7 and characterization by x-ray absorption and fourier transform infrared spectroscopy. J. Cryst. Growth 92, 239–252 (1988).
Harries, J. E., Hukins, D. W. L., Holt, C. & Hasnain, S. S. Conversion of amorphous calcium phosphate into hydroxyapatite investigated by EXAFS spectroscopy. J. Cryst. Growth 84, 563–570 (1987).
Larson, D. J., Prosa, T. J., Ulfig, R. M., Geiser, B. P. & Kelly, T. F. Local Electrode Atom Probe Tomography: A User’s Guide (Springer Science and Business Media, 2013).
Thompson, K. et al. In situ site-specific specimen preparation for atom probe tomography. Ultramicroscopy 107, 131–139 (2007).
Qi, M.-l., Xiao, G.-y. & Lu, Y.-p. Rapid hydrothermal synthesis of submillimeter ultralong flexible hydroxyapatite fiber using different pH regulators. Acta Metall. Sinica Engl. Lett. 29, 609–613 (2016).
Dolomanov, O. V., Bourhis, L. J., Gildea, R. J., Howard, J. A. K. & Puschmann, H. OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 42, 339–341 (2009).
Sheldrick, G. M. Crystal structure refinement with SHELXL. Acta Crystallogr. C 71, 3–8 (2015).
Perdew, J. P. et al. Restoring the density-gradient expansion for exchange in solids and surfaces. Phys. Rev. Lett. 100, 136406 (2008).
Giannozzi, P. et al. QUANTUM EXPRESSO: a modular and open-source software project for quantum simulations of materials. J. Phys. Condens. Matter 21, 395502 (2009).
Vanderbilt, D. Soft self-consistent pseudopotentials in a generalized eigenvalue formalism. Phys. Rev. B 41, 7892–7895 (1990).
Dal Corso, A. Pseudopotentials periodic table: from H to Pu. Comput. Mater. Sci 95, 337–350 (2014).
Babushkin, O., Lindbäck, T., Holmgren, A., Li, J. & Hermansson, L. Thermal expansion of hot isostatically pressed hydroxyapatite. J. Mater. Chem. 4, 413–415 (1994).
Rohatgi, A. WebPlotDigitizer Version 4.2 https://automeris.io/WebPlotDigitizer (2019).
Acknowledgements
This work was supported in part by the National Institute of Health–National Institute of Dental and Craniofacial Research (NIH-NIDCR R03 DE025303-01, R01 DE025702-01), the National Science Foundation (DMR-1508399), the NSF Platform for the Accelerated Realization, Analysis and Discovery of Interface Materials (PARADIM) under Cooperative Agreement no. DMR-1539918, and the University of Virginia. K.A.D. was in part supported by a 3M fellowship. The Canadian National Sciences and Engineering Research Council in part supported L.M.G. K.A.D. and M.J.C. were supported in part by the Northwestern University Graduate School Cluster in Biotechnology, Systems and Synthetic Biology, which is affiliated with the Biotechnology Training Program. L.S. was supported by a Deutsche Forschungsgemeinschaft research fellowship (STE2689/1-1). This work made use of the following core facilities operated by Northwestern University: NUCAPT, which received support from NSF (DMR-0420532), ONR (N00014-0400798, N00014-0610539, N00014-0910781 and N00014-1712870), and the Initiative for Sustainability and Energy at Northwestern University (ISEN); MatCI; NUANCE and EPIC, which received support from the International Institute for Nanotechnology (IIN), the Keck Foundation, and the State of Illinois, through the IIN; IMSERC; the Jerome B. Cohen X-Ray Diffraction Facility; QBIC, which received support from NASA Ames Research Center (NNA06CB93G). NUCAPT, MatCI, NUANCE and EPIC were further supported by the MRSEC programme (NSF DMR-1720139) at the Materials Research Center; NUCAPT, NUANCE, EPIC and IMSERC were also supported by the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205). This work made use of the Cornell Center for Materials Research (CCMR) Shared Facilities supported through the NSF MRSEC Program (no. DMR-1719875). The Titan Themis 300 was acquired through NSF-MRI-1429155, with additional support from Cornell University, the Weill Institute, and the Kavli Institute at Cornell. This work made use of the Rivanna cluster maintained by the Advanced Research Computing Services at the University of Virginia. Portions of this work were performed at the Canadian Light Source (CLS), which received support from The Natural Sciences and Engineering Research Council of Canada, the National Research Council of Canada, the Canadian Institutes of Health Research, the Province of Saskatchewan, Western Economic Diversification Canada, and the University of Saskatchewan. The authors thank P. Akers, M. Stohle and G. Borden for providing de-identified human premolars, and C. Malliakas, K. MacRenaris, M. Thomas and especially K. Rice for technical support.
Author information
Authors and Affiliations
Contributions
The experiments and simulations were designed by K.A.D., P.J.M.S., L.S., P.V.B., J.M.R., D.J. STEM experiments were performed by P.J.M.S., B.H.G. and M.J.Z., with additional help in analysis and simulations provided by M.J.Z. and L.F.K. APT data were collected and analysed by K.A.D., with D.J. assisting the analysis. The finite-element model was developed by K.A.D. and D.J. using DFT calculations performed by P.V.B., and synthetic Mg-rich OHAp crystals made and analysed by L.S. XAS data were collected and analysed by L.M.G. and M.J.C. K.A.D., P.J.M.S., B.H.G., P.V.B., M.J.Z., L.F.K., J.M.R. and D.J. were all involved in preparing the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Supplementary Information
This file contains Supplementary Methods, Tables, Discussion, and References, organized by experimental technique (1: STEM; 2: XAS; 3: APT; 4: FEM; 5: SEM) with an additional chapter that details the proposed model for amelogenesis in greater detail. This section contains 21 Supplementary Figures, 9 Supplementary Tables, and 40 references.
Supplementary Data
This zipped file contains source data for Supplementary Figures 4–7 and 14–17.
Video 1
Ion positions in a human enamel crystallite. All 24Mg2+ (magenta), 40Ca19F+ (blue), 23Na+ (turquoise), and COxHy+ ions (red) are rendered for a 30 nm thick slice through a human enamel crystallite (crystallite D in Supplementary Fig. S10a). Note that dataset was rotated such that view direction (x axis) is approximately parallel to the Mg layers. The animation shows rotation about the z axis, which is normal to the Mg layers. This visualization was scripted using the Matlab R2019b platform (The MathWorks).
Video 2
Core-shell and sandwich structure of human enamel crystallite. The reconstruction was rotated as in Video 1, voxelized (1 nm isotropic voxels) and smoothed. Iso-concentration surfaces and iso-caps for 24Mg2+ (magenta, ciso = 0.1 nm−3), 23Na+ (turquoise, 0.3 nm−3), and COxHy+ ions (red, 0.12 nm−3) were rendered together with that for 40Ca19F+ (blue, 0.07 nm−3) for the same slice as in Video 1, but ions outside the central crystallite are not shown. The animation shows one revolution about the z axis, followed by an oscillatory tilt series about the y axis. This visualization was scripted using the Matlab R2019b platform (The MathWorks).
Rights and permissions
About this article
Cite this article
DeRocher, K.A., Smeets, P.J.M., Goodge, B.H. et al. Chemical gradients in human enamel crystallites. Nature 583, 66–71 (2020). https://doi.org/10.1038/s41586-020-2433-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-020-2433-3
This article is cited by
-
Biomimetic Construction of the Enamel-like Hierarchical Structure
Chemical Research in Chinese Universities (2023)
-
Magnesium stable isotope composition, but not concentration, responds to obesity and early insulin-resistant conditions in minipig
Scientific Reports (2022)
-
Restoration of enamel anti-wear properties via remineralization: Role of occlusal loading
Friction (2022)
-
Strong and Tough TPU Fibers with Orientedly Aligned CNTs Reinforced by Amorphous ZrO2
Chemical Research in Chinese Universities (2022)
-
Advances in biomineralization-inspired materials for hard tissue repair
International Journal of Oral Science (2021)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.