Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 µm for in vivo imaging to 1.5 µm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium-carbon ratio. However, the methods mentioned above only show the result-already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels-starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high-resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: In vivo MicroCT scanning of mouse Basic Protocol 2: Ex vivo HR-MicroCT of the teeth Basic Protocol 3: SEM for teeth microstructure Basic Protocol 4: Stimulated emission depletion (STED) microscopy.

Czech Centre for Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Imaging Methods Core Facility Charles University Prague Faculty of Science Prague Czech Republic

Laboratory of Transgenic Models of Diseases Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Boyde, A. (1989). Enamel. In Teeth (pp. 309-473). Berlin, Heidelberg: Springer. doi: 10.1007/978-3-642-83496-7_6.

Delgado, S., Ishiyama, M., & Sire, J.-Y. (2007). Validation of amelogenesis imperfecta inferred from amelogenin evolution. Journal of Dental Research, 864, 326-330. doi: 10.1177/154405910708600405.

Donovan, J., & Brown, P. (2006). Parenteral injections. Current Protocols in Immunology, 73, 1.6.1-1.6.10. doi: 10.1002/0471142735.im0106s73.

Dumbryte, I., Vailionis, A., Skliutas, E., Juodkazis, S., & Malinauskas, M. (2021). Three-dimensional non-destructive visualization of teeth enamel microcracks using X-ray micro-computed tomography. Science Reports, 11, 14810. doi: 10.1038/s41598-021-94303-4.

Gibson, C. W., Yuan, Z. A., Hall, B., Longenecker, G., Chen, E., … Kulkarni, A. B. (2001). Amelogenin-deficient mice display an amelogenesis imperfecta phenotype. The Journal of Biological Chemistry, 276(34), 31871-31875. doi: 10.1074/jbc.M104624200.

Hu, J.C-C., Hu, Y., Lu, Y., Smith, C. E., Lertlam, R., … Simmer, J. P. (2014). Enamelin is critical for ameloblast integrity and enamel ultrastructure formation. PLoS ONE, 9(3), e89303. doi: 10.1371/journal.pone.0089303.

Kapila, S. N., Natarajan, S., Boaz, K., Pandya, J. A., & Yinti, S. R. (2015). Driving the mineral out faster: Simple modifications of the decalcification technique. Journal of Clinical and Diagnostic Research, 9(9), ZC93-ZC97. doi: 10.7860/JCDR/2015/14641.6569.

Lacruz, R. S., Habelitz, S., Wright, J. T., & Paine, M. L. (2017). Dental enamel formation and implications for oral health and disease. Physiological Reviews, 97, 939-993. doi: 10.1152/physrev.00030.2016.

Mazumder, P., Prajapati, S., Bapat, R., & Moradian-Oldak, J. (2016). Amelogenin-ameloblastin spatial interaction around maturing enamel rods. Journal of Dental Research, 95(9), 1042-1048. doi: 10.1177/0022034516645389.

Møinichen, C. B., Lyngstadaas, S. P., & Risnes, S. (1996). Morphological characteristics of mouse incisor enamel. Journal of Anatomy, 189(Pt 2), 325-333.

Schmitz, J. E., Teepe, J. D., Hu, Y., Smith, C. E., Fajardo, R. J., & Chun, Y. H. (2014). Estimating mineral changes in enamel formation by ashing/BSE and microCT. Journal of Dental Research, 93(3), 256-262. doi: 10.1177/0022034513520548.

Smith, C. E., & Nanci, A. (1995). Overview of morphological changes in enamel organ cells associated with major events in amelogenesis. International Journal of Developmental Biology, 39, 153-161.

Smith, C. E. L., Poulter, J. A., Antanaviciute, A., Kirkham, J., Brookes, S. J., … Mighell, A. J. (2017). Amelogenesis imperfecta; genes, proteins, and pathways. Frontiers in Physiology, 8, 435. doi: 10.3389/fphys.2017.00435.

Wald, T., Spoutil, F., Osickova, A., Prochazkova, M., Benada, O., … Osicka, R. (2017). Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif. Proceedings of the National Academy of Science, 114(9), E1641-E1650. doi: 10.1073/pnas.1615334114.

Wazen, R. M., Moffatt, P., Zalzal, S. F., Yamada, Y., & Nanci, A. (2009). A mouse model expressing a truncated form of ameloblastin exhibits dental and junctional epithelium defects. Matrix Biology, 28(5), 292-303. doi: 10.1016/j.matbio.2009.04.004.

Wood, C. B., & Stern, D. N. (1997). The earliest prisms in mammalian and reptilian enamel. In W. Koenigswald & P. M. Sander (Eds.). Tooth enamel microstructure. (pp. 63-83). Rotterdam: A. A. Balkema.

Zeller, R. (2001). Fixation, embedding and sectioning of tissues, embryos, and single cells. Current Protocols in Pharmacology, 7, A.3D.1-A.3D.9. doi: 10.1002/0471141755.pha03ds07.