Revealing Dentin Multiscale Structures Using High-Resolution Transmission Electron Microscopy

Like bone, dentin exhibits a complex multiscale hierarchical structure. Dentin microstructure has been widely studied at the microscale, using direct 2-dimensional observation techniques such as optical and scanning electron microscopy. Its porous network microstructure has been recently revealed in 3 dimensions 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 electron microscopy (TEM) and selected area electron diffraction to analyze the 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 CF orientation and crystal shapes. The study concludes with the following outcomes: (1) the entanglement of CFs and hydroxyapatite minerals is revealed as a woven structure with various degrees of isotropy from the middle dentin to the sub-DEJ zone, (2) individual HAP crystals are elongated along their 3 crystal directions (a, b, and c axes) and assembled to create S-shaped structures around adjacent CFs, and (3) the yet-unknown transition zone between peritubular dentin 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.