# Revealing Dentin Multiscale Structures Using High-Resolution Transmission Electron Microscopy

Margot Leclercq, Maxime Vallet, Thomas Reiss, Ariane Berdal, Elsa Vennat

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## To cite this version:

Margot Leclercq, Maxime Vallet, Thomas Reiss, Ariane Berdal, Elsa Vennat. Revealing Dentin Multiscale Structures Using High-Resolution Transmission Electron Microscopy. Journal of Dental Research, 2025, 10.1177/00220345251361912. hal-05236363

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# Revealing dentin multiscale structures using High Resolution Transmission Electron Microscopy (HR-TEM)

Margot Leclercq<fnref n="1" />*, Maxime Vallet<fnref n="1" />,<fnref n="2" />, Thomas Reiss<fnref n="1" />, Ariane Berdal<fnref n="3" />,<fnref n="4" />, Elsa Vennat<fnref n="1" />*

## Abstract

Like bone, dentin exhibits a complex multi-scale hierarchical structure. Dentin microstructure has been widely studied at the microscale, using direct 2D observation techniques such as optical and scanning electron microscopy. Its porous network microstructure has been recently revealed in 3D using confocal laser scanning microscopy. However, at the nanoscale, the organization of collagen fibrils (CFs) and mineral crystals in the dentinal tissue remains unclear. This study uses High-Resolution Transmission Electronic Microscopy (HR-TEM) and Selected Area Electron Diffraction (SAED) to analyze dentin nanostructure. Two areas are discussed, one near the dentin-enamel junction (DEJ) and the other in the middle dentin. Special attention is paid to the TEM section orientation to the tubule axis for discussing the CFs orientation and crystal shapes. The study concludes with the following outcomes:

- CFs and HAP minerals entanglement is revealed as a woven structure with various degrees of isotropy from the middle dentin to the sub-DEJ zone.

- Individual HAP crystals are elongated along their three crystal directions (a, b, and c axes) and assembled to create S-shaped structures around adjacent CFs.

- The yet unknown transition zone between peritubular dentin (PTD) and intertubular dentin (ITD) comprises CFs parallel to the tubule axis, which could explain the crack propagation from the tubules to the ITD.

This study proposes a protocol for selecting and collecting a TEM dentin sample at a specific depth and orientation. This method enables the investigation of mineral/organic entanglement in dentin at various locations within the same tooth, paving the way for a better understanding of structural gradients and the structure-properties relationship of the dentinal tissue.

**Keywords:** Dentin nanostructure, High Resolution Transmission Electron Microscopy (HR-TEM), Collagen fibrils / HAP crystals organization, Structure / properties relationship.

## 1. Introduction

Dentin tissue is a natural composite made up of a mineral phase of carbonated hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) crystals (HAP), approximately 50% by volume, an organic matrix composed mainly of

<footnote n="1">¹ Université Paris-Saclay, CentraleSupélec, ENS Paris-Saclay, CNRS, LMPS - Laboratoire de Mécanique Paris-Saclay, 91190, Gif-sur-Yvette, France.</footnote>

<footnote n="2">² Université Paris-Saclay, CentraleSupélec, CNRS, Laboratoire Structures, Propriétés et Modélisation des solides (SPMS), 91190, Gif-sur-Yvette, France.</footnote>

<footnote n="3">³ Laboratoire de Pathophysiologie Orale Moléculaire, Centre de Recherche des Cordeliers, INSERM UMRS, Université Paris Cité, Sorbonne Université, Paris 1138, France</footnote>

<footnote n="4">⁴ Fédération Hospitalo-Universitaire DDS-ParisNet, INSERM, Université Paris Cité, Assistance Publique-Hôpitaux de Paris, France</footnote>

<footnote>*Corresponding authors: margot.leclercq@centralesupelec.fr (M. Leclercq); elsa.vennat@centralesupelec.fr (E. Vennat)</footnote>
type I collagen, approximately 30% by volume, and water (Kinney et al., 2003). Its degree of mineralization is comparable to the one of bone tissue (Vennat, 2009). On a microscopic scale, the dentin matrix is composed of a core of Intertubular Dentin (ITD) containing truncated cone-shaped pores (lumen) reinforced by a highly mineralized peritubular collar called peritubular dentin (PTD). This PTD + lumen assembly is called a tubule and has an internal diameter ranging from 0.8 to 2.5 µm depending on the location in dentin (Marshall et al., 1997), (Vennat et al., 2021). The tubules fan out from the pulp-dentin junction to the dentin-enamel junction (DEJ) in the crown and the cemento-dentin junction in the root (Tjäderhane et al., 2009). Several studies observed a significant anisotropy of the dentinal tissue in relation to its fracture properties (Iwamoto and Ruse, 2003) (Rasmussen and Patchin, 1984). However, Kinney et al. shows that the tubule orientation had no effect on the elastic behavior of the tissue (Kinney et al., 1996). Hence, the observed anisotropic properties of the dentin tissue were suggested to be underlied by its nanostructure. Currently, its nanostructure is still debated and only few studies discussed the structure - properties link at this scale level. ITD is located between the tubules and is composed of an entanglement of type I collagen fibrils (CFs) and HAP crystals. The CFs are characterized by gap regions and overlapping areas of several tropocollagen molecules. This organization leads to a periodicity called D-banding of 67 nm, characteristic of CFs (Petruska and Hodge, 1964). Several studies have shown that CFs are predominantly oriented in planes perpendicular to the longitudinal axis of the tubules in the middle dentine. However, authors disagree on fibrils orientation and anisotropy in the mantle dentin at the DEJ and in the predentin at the pulp-dentinal junction (Kinney et al., 2001) (Elbaum et al., 2007).

Concerning the mineral phase, ITD has two types of HAP crystals: the extra-fibrillar and intra-fibrillar ones, forming the inorganic phase of this extracellular matrix. Intra-fibrillar minerals are mainly confined to intermolecular gap area, created by the tropocollagen molecular arrangement. They enhance the periodic contrast observed by Transmission Electron Microscopy (TEM) in the CFs (Argunova et al., 2020). The extra-fibrillar crystals are organized between the fibrils as gathered clusters. Their size and shape pattern in the different layers of dentine is discussed in several studies (Kinney et al., 2001) (Xue et al., 2013) (Tesch et al., 2001). Iwamoto and Ruse studied fracture behavior of dentin tissue and concluded that the fracture toughness may be dependent on the degree of mineralization of dentin (Iwamoto and Ruse, 2003). Hence, clarifying HAP / CFs entanglement at the nanoscale level is required to understand the influence of this nanostructure on the mechanical properties of the tissue.

Another interesting area is the junction zone between ITD and PTD. The structural organization of this region was studied by demineralizing the dentin to reveal its organic network (Cohen et al., 2008), (Vennat, 2009). Several authors reported an organic membrane, known as the *lamina limitans*, made up of proteoglycans and glycosaminoglycans which lies at this junction (Bertassoni, 2017). Some others referred to this area as the ‘transition zone’ where the local elastic modulus decreases from the PTD modulus to reach the ITD modulus (Balooch et al., 2004), (Perdigao et al., 1995). Few links are made between structure and mechanical properties which is nonetheless crucial for understanding crack propagation behavior at the interface between ITD and PTD.

Most existing mechanical models do not consider the nanostructure of the dentin tissue, whereas the CP/HAP crystal interlocking may represent the main source of the anisotropic properties observed in dentin (Zaslansky et al., 2006). This paper aims to give a better description of the dentin nanostructure by combining Focused Ion Beam (FIB), for selection of regions of interest, with HR-TEM to investigate its nanostructure and link it to the known mechanical properties of the tissue.
**2. Materials and Methods**

**2.1. Sample Preparation**

A permanent molar of a young patient without visible pathological signs was obtained with donor consent. The molar was first stored in 0.5% Chloramine T and then in Ringer liquid at 5°C. The tooth has been embedded in EpoFix resin. Then, it was cut from crown to root with a diamond-edged slow-speed saw to ensure that cross-section surfaces were approximately parallel to the tooth's longitudinal plane or to the dentinal tubules' longitudinal axis. Both surfaces were mechanically polished using abrasive Si-C paper and then cleaned with ultrasounds to remove any residue. The final polishing stage involves fine granulometry paper while lubricating with a diamond solution. The sample was progressively dehydrated through increasing ethanol concentrations up to 100%. Samples were then fixed to a flat pin stub with conductive silver paint. A few nm thick layer of gold was finally deposited on the sample surface.

**2.2. Focused Ion Beam Milling**

Thin sections were prepared using FEI ThermoFisher Helios Nanolab 660 focused ion beam – scanning electron microscope equipped with a 30 kV gallium ion column, gas injector system, and a NanoManipulator system EasyLift for TEM sample in situ lift-out. Such preparation method was shown to produce high-quality TEM sections with minor structural artefacts (Witzke et al., 2022). Specimens were thinned until approximately 100 nm to obtain electron transparency using a current reduction from 0.79 nA to 80 pA at 30 kV. The sections were cut in two orientations: parallel to the orientation of the long axis of the tubules (//) and perpendicular or transverse to the long axis of tubules (⊥) and at two locations: one near DEJ (sub_DEJ) and the second in middle dentin (M) as illustrated in Supplementary Table 1.

**2.3. Transmission Electron Microscopy**

Sections prepared by conventional FIB-SEM procedure were then observed with a FEI ThermoFisher TITAN³ G2 80-300 TEM equipped with a Cs probe corrector given a 0.07 nm spatial resolution. Images were acquired at 300 kV by coupling high-angle annular dark-field scanning TEM (HAADF STEM), conventional bright-field TEM (BF TEM), selected area electron diffraction (SAED) as well as high resolution TEM (HR TEM).

**2.4. Image Analysis**

The TEM image analysis was done using the ImageJ Fiji software (Schindelin et al., 2012). The curtaining effect due to SEM-FIB for TEM sections preparation was reduced using the BandPass Filter tool. The orientation of HAP crystals was evaluated using the OrientationJ plugin.

**3. Results and Discussion**

**3.1. Middle dentin: from micro- to nanostructure**

**3.1.1. Collagen fibrils tridimensional organization**

Figure 1 presents images of the section perpendicular to the tubule extracted from the middle dentin area ($P_{⊥}^{M}$). Around the lumen of the tubule on the left side of the image, the dense collar of PTD ranged from 100 nm in some areas to 350 nm thick. This PTD collar did not present any CFs or any pore, consistently
with reported observations (Xu and Wang, 2012) (Weiner et al., 1999) (Habelitz et al., 2007). Over a large
majority of the ITD section, an entanglement of type I CF and apatite minerals was visible. The HAP crystals
in the HAADF STEM mode appear as brighter elongated structure. A large majority of CFs lied in the plane
of the section, characterized by alternating dark and bright stripes along their length (Fig 1A). Figure 1B
shows a HAADF STEM image of the transition zone between PTD and ITD. This interface was composed
of fibrils mainly oriented along the tubule lumen and highly intra-mineralized. CFs appeared to be
progressively aligned from parallel to perpendicular to the tubule axis with less dense intra-mineralization
as one moves away from the PTD.

This transition zone where the fibrils were shown here to gradually change their orientation is a poorly studied although it links two areas with very different properties: a rigid PTD and more flexible ITD. As the dense collars of PTD is exposed to high stress concentration, they are prone to crack. However, it had been seen on dehydrated dentin that cracks do not naturally propagate in ITD (Vennat et al., 2021). This might be explained by those longitudinally oriented mineralized fibers creating a crack deflection effect. Also, it is an intermediary area avoiding sharp changes in local properties and providing a smooth interface with a rigidity gradient between PTD and ITD. These conclusions are consistent with previous studies on dentin elastic behavior and fractures properties (Iwamoto and Ruse, 2003; Kinney et al., 1996). Here, this transition zone was extending over 900 nm in some area of the section, which is consistent with reported values (Balooch et al., 2004), (Cohen et al., 2008), (Xu and Wang, 2012). The average orientation of CFs was studied here and is shown in Figure 1C. The whole section analysis revealed a woven structure, with successive superposition of several bundles of CFs and extra-mineral oriented in different directions, in good agreement with Kinney et al. (Kinney et al., 2001). A schematic representation of the discussed organization of mineralized CFs from PTD to ITD is proposed in Supplementary Figure 1.

Figure 1D and 1E shows elongated structure of HAP crystals (in black) that appeared to be oriented along the CF and its corresponding HSB color-coded map for orientation analysis in $P_{\perp}^M$. The angle distribution shows that crystals were mainly oriented in the direction of the CF with a small angle dispersion. The angle dispersion of crystals around the CF creates a “S-shape” structure of HAP crystals around the CF. The color-coded map reveals this “S-shape” of crystal structures which appeared to be formed by several individual crystals, allowing the crystalline structure to curve with such an angle around the CF. Figure 1F is a diffraction pattern of the area surrounded in orange in Fig 1D, showing rings that correspond to the characteristic hkl reflections of HAP crystals obtained by Selected Area Electron Diffraction (SAED) in $P_{\perp}^M$. The two bright spots (white arrow) are reflections of plans (002). No continuous ring is visible for those plans which confirm the preferential orientation of the crystals, having their c-axis forming the main axis of the crystalline structures aligned in the same direction. Supplementary Figure 2 presents the section parallel to the longitudinal axis of tubules in middle dentine ($P_{\parallel}^M$). Collagen fibrils were lying mainly in planes roughly perpendicular to the cross-sectional plane, i.e., they lied perpendicular to the tubules' longitudinal axis. The associated diffraction patterns showed that HAP crystals are mainly aligned with their c-axis parallel to the CF, which is consistent with analysis made on $P_{\perp}^M$ and previous studies (Argunova et al., 2020).

### 3.1.2.Collagen fibrils – crystals local entanglement

The figure 2 focuses on a single CF of the section $P_{\perp}^M$ and the orientation of HAP crystals in this specific area. SAED and HRTEM coming from the same area are shown in Fig 2B and 2D. The first ring, corresponding to (002) plane-related reflection, was not continuous and formed two arcs with an angular
dispersion aligned around the main axis of the CF similarly to what was observed in Figure 1. Figures 2C and 2D are HR-TEM images showing atomic arrangement of plans (002) of 3 individual crystals with its Fast Fourier Transform (FFT) and one crystal, respectively. Bright spots in the FFT correspond to plans (002) of each HAP crystal shown in 2A, which gives each crystal c-axis orientation. These crystals show an elongated shape preferentially oriented along their a- and b-axis. This figure also shows the angular dispersion of these crystals along the direction of the fibril, giving information on the orientation of individual crystals in the plane.

These findings on individual crystals might offer an explanation for the overall “S-shaped” crystalline structure previously described in section $P_{\perp}^M$. The crystalline formations consist of an assembly of numerous individual crystals oriented in various directions, enabling the curvature reported previously on dentin and bone (Grandfield et al., 2018), (Xue et al., 2013), (Reznikov et al., 2018). Grandfield et al. described two different types of clusters of HAP crystals in cortical bone: curved “mineral lamellae” (ML) wrapping locally around CFs and flattened ML between CFs that becomes increasingly flat with distance to the CF (Grandfield et al., 2018). In dentin, such flat and straight stacks of crystalline structures between CFs are not observed. The crystals shapes and organization in dentin layers seem to be closer to the cortical bone structural model of Reznikov et al. (Reznikov et al., 2018). In the proposed model, individual HAP crystals are merging to form “S-shaped” crystalline structures, roughly parallel to one another around adjacent CFs which could significantly contributes to the structural integrity of dentin tissue (see Supplementary Figure 5).

## 3.2. Sub-DEJ dentin: differences in micro- and nanoscale structures and discussion on tissue's mechanics

### 3.2.1. Sub-DEJ dentin microstructure: tubules and lateral branches

Figure 3 shows dentin tissue in the $P_{\perp}^{sub\_DEJ}$ section. A thin PTD collar with a thickness of 15–20 nm was surrounding the tubule lumen. The PTD collar was thinner in sub-DEJ dentin compared to middle dentin, as described in previous studies (Zaslansky et al., 2006) (Pashley, 1996). The transition zone between PTD and ITD was also visible in the sub-DEJ region, around 400–600 nm thick. Such dimension of the transition zone is consistent with previous studies, describing diameters of transition area between 300 and 1000 nm (Balooch et al., 2004), (Cohen et al., 2008), (Xu and Wang, 2012). On the section $P_{\parallel}^{sub\_DEJ}$ (Figure 4) we observed a lateral branch (“LB”) with a diameter of 230 nm surrounded by a dense collar probably arising from the tubule. This range of diameter agrees with previous observations of LB under SEM made by several authors (Mjör and Nordahl, 1996) (Vennat et al., 2017), (Vennat et al., 2021). It has not the same appearance that the PTD collar in which appears uniformly white on HAADF-STEM images. The LB collar presents bundles of a tenth of HAP lying radially with respect to the LB lumen.

### 3.2.2. Collagen fibrils - HAP crystals in dentin layers: impact on tissue mechanics

To compare fibril's directions in sub-DEJ and middle dentin sections, HSB color-coded map and corresponding angle distribution are presented in Figure 3 ($P_{\perp}^{DEJ}$). The woven structure is not clearly visible in the map, as only single CFs are distributed with a large angle in the entire section, showing a relative in-plane isotropy. To compare the degree of isotropy between the sections, a criterion was defined as the number of CFs intersecting the plane of the section - i.e., not aligned in the plane - per unit area. Approximately 8 CFs/µm² are not lying in the plane compared to only 1.5 CFs/µm² for the middle dentine
section. At 10 – 30 µm beneath the DEJ, CFs are reported as changing their orientation from perpendicular to parallel to the DEJ, i.e., parallel to perpendicular to tubules (Moss, 1974; Ten Cate, 1978). Zaslansky et al. shows fibrils in all orientations relative to the tubule at a distance of 20 - 50 µm from DEJ and fibrils with preferential orientation in the incremental growth plane deeper in the crown, at ≈ 800 µm (Zaslansky et al., 2006). Our results suggest that the organization of CFs from DEJ to middle dentine is very gradual and that the transition could extend over several hundred µm. A schematic representation of the supposed collagen fibrils (CFs) organization from enamel to bulk dentin is proposed in Figure 5. This organization in middle dentin confers to dentin a lower toughness when the load tends to separate the parallel planes where the fibrils are lying (Iwamoto and Ruse, 2003). In the other direction the crack is more difficult to propagate since fibrils are prone to bridge the crack path. It can explain why the cracks are stopped below DEJ and not at DEJ since the nanostructure directly below DEJ will have less fibrils playing this crack-arresting role (Imbeni et al., 2005). We can also infer that the more anisotropic organization near DEJ has a mechanical role to play in shear stress bearing, in complement with the scalloped interface between dentin and enamel. Chan et al. concluded that it is the presence of CFs crossing the DEJ that is responsible for the low delamination probability of the DEJ, reinforcing the hypothesis of the necessity of the organization described in the Figure 5 (Chan et al., 2011).

### 3.3. Method limitations and perspectives

Due to the experimental methods, the presented results are limited to observing a single tooth (molar) in regions of a few microns. The drawn conclusions are valid for the conditions of the studied tooth, as its mechanical behavior is known to be impacted by conditions such as aging (Arola and Reprogel, 2005). The proposed approach is useful for studying variability between tooth types, and could be extended to animal kingdom, subject to other types of mechanical stress (Bertassoni, 2017; Ho et al., 2009). Additional observations were made on another healthy where the exacts location of the section concerning the DEJ is unknown. However, the CFs and S-shaped HAP crystals entanglement is in agreement with the proposed model (Supplementary image 7.A). At the PTD – ITD junction level, mineralized CFs parallel to the main axis of the tubule are also visible forming the gradually mineralized PTD – ITD junction as a sheath. Hence, the very local organization of dentin nanostructure was highlighted. Like the DEJ area at the tissue level (Imbeni et al., 2005), the PTD – ITD junction raises questions regarding the mechanical role of the interface (An and Wagner, 2017). Studying the mechanical behavior of this junction by electron microscopy would provide a better understanding of the mechanical impact of such a natural interface on the tissue.

This study could shed new light on previous macro-scale results concerning the mechanical properties of dentin by linking larger-scale phenomena with nano-scale structure. In particular, Arola and Reprogel showed that dentin's fracture path depended on the tubules' orientation and, thus, the CFs planes (Arola and Reprogel, 2006). Furthermore, Ivancik and Arola inferred that dentin's fracture toughness depended on the tissue's depth, which implies a different nano-scale arrangement, as shown in the current study (Ivancik and Arola, 2013).

These results add to the pre-existing literature on dentin multiscale structures, confirming a nanostructure gradient and the microstructure gradient already established within dentin. To our knowledge, this study is the first to propose a HAP – CFs entanglement model for dentinal tissue which could lead to new insights into its structure-properties link.

## 4. Conclusion
Dentin is a complex biological hard tissue presenting a hierarchical organization at different scales. At the nanoscale, dentin shows an organization involving a mineral phase made of HAP crystals and an organic phase mainly made of CFs. In this article, we used HR-TEM and SAED to bring new insight into this ultrastructure. By FIB milling we ensured our TEM sections to be either parallel or perpendicular to the tubular path. We investigated dentin ultrastructure near DEJ and in middle dentin and we showed that:

- The CFs are organized in a more isotropic way near DEJ than in middle dentin where they are lying in planes perpendicular to the tubule axis. In those planes perpendicular to tubule axis, bundles of CFs and mineral are oriented in preferential directions, creating a woven structure.

- The crystals are:

* Organized in S-shaped crystalline structure of merged individual HAP crystals with various orientation around adjacent CFs.

* Elongated mainly along their c-axis but were also seen elongated in the other crystalline directions.

- The transition zone between PTD and ITD is made of mineralized CFs running parallel to the tubule axis, unlike in the rest of ITD. This observation might explain the behavior of cracks initiated in tubules, that do not propagate in ITD.

## Authors contribution

Margot Leclercq contributed to conception, design, data acquisition and interpretation, and drafted the manuscript. Maxime Vallet contributed to data acquisition, analysis and interpretation and critically revised the manuscript. Thomas Reiss contributed to conception, design, data acquisition and interpretation and critically revised the manuscript. Ariane Berdal contributed to interpretation and critically revised the manuscript. Elsa Vennat contributed to conception, design, data analysis and interpretation and critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.

## Funding acknowledgments

This work benefited from the financial support of the LabeX LaSIPS (ANR-10-LABX-0040-LaSIPS) managed by the French National Research Agency under the ‘Investissements d'avenir’ program (ANR-11-IDEX-0003-02) and was carried out within the MATMECA consortium and supported by the ANR under contract number ANR-10-EQPX-37.

## Conflict-of-interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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List of figures

**Figure 1.** (A) Image stitching of section $P_{\perp}^M$ in HAADF-STEM mode. In intertubular dentin, collagen fibrils are oriented in section's plane, visible by the characteristic ≈ 67-nm space banding of these fibrils. Some fibrils are oriented in planes normal to the section (black holes); scale bar = 200 nm. (B). Collagen fibril in planes normal to the section surrounded by bundle of minerals at the transition zone between peritubular and intertubular dentin (double arrow); scale bar = 50 nm. (C) HSB color-coded map obtained with OrientationJ analysis (Gaussian window = 6x6 pixels) and its corresponding polar coordinate histogram: orientation = hue, coherency = saturation (D) Bright-field TEM image of mineral (black) surrounding a collagen fibril (surrounded in red) in intertubular dentin on $P_{\perp}^M$ section. (E) HSB color-coded map obtained with OrientationJ analysis and its corresponding polar coordinate histogram (Gaussian window = 6x6 pixels): orientation = hue, coherency = saturation; scale bar = 50 nm (F) Diffraction pattern of the area surrounded in orange in (D); scale bar = 10 nm⁻¹.

**Figure 2.** (A) Bright-Field (BF) TEM image of a CF in the plane (surrounded in red), main axis of the CF is represented by the yellow arrow; scale bar = 500 nm. (B) Selected Area Electron Diffraction (SAED) obtained on the yellow circle inside the fibril visible in (A). The blue area shows angle dispersion of plans (002) around the CF axis. (C) HR-TEM image inside the yellow circle and its Fast Fourier Transform (FFT). Color inverse FFT showing plans (002) of crystals oriented with different angles around the CF axis; scale = 10 nm. (D) HR-TEM image inside a single crystal, showing arrangement of atomic plans (002) along its long axis.

**Figure 3.** (A) Image stitching of section $P_{\parallel}^{sub\_DEJ}$ in HAADF-STEM mode; scale bar = 200 nm. (B) HSB color-coded map obtained with OrientationJ analysis (Gaussian window = 6x6 pixels) and its corresponding angle distribution in the section surrounded in yellow in A: orientation = hue, coherency = saturation.

**Figure 4.** Images stitching of section $P_{\parallel}^{sub\_DEJ}$ in HAADF-STEM mode, one CF in the plane of the section is surrounded in red, LB = Lateral branch; scale bar = 200 nm.

**Figure 5.** Schematic view of the supposed collagen fibrils (CFs) organization from enamel to bulk dentin and its consequences on the mechanics of the tissue.