New insights into nanoscale organization of dentin

Dentin is the main tissue of the tooth, located between the enamel and the pulpal cavity. Dentin displays a hierarchical organization with microscale porosities surrounded by a dense mineral collar (peritubular dentin, PTD) called tubules, embedded in a matrix of intertubular dentin (ITD). At the nanoscale, the ITD matrix is made up of an entanglement of collagen fibrils (roughly 100nm diameter) and mineral platelets (roughly 5nm thick). X-Ray ptychotomography (PXCT), a recent nano-tomography technique, has already been used by Zanette et al. to analyze the dentin nanostructure, reaching a resolution of about 160nm, superior to the collagen fibril diameter. We present here a new PXCT study with a resolution of 50nm. Two healthy teeth were examined, one permanent and one primary. The samples were cut and polished to the micron using classical techniques. Cylindrical samples of roughly 20 microns in height and 20 microns in diameter were extracted, with a first step of coarse micromachining using a Xenon Plasma FIB-SEM (TESCAN FERA 3), followed by the extraction and fixation of the sample on the pin, using a Gallium FIB-SEM (Helios 660, FEI). The PXCT experiments were performed at Synchrotron Soleil on the SWING beamline. A coherent X-ray beam with an energy of 8 keV was focused ahead of the sample position, resulting in a 4 µm probe size. For each tomographic projection, about 500 diffraction patterns were recorded with an exposure time of 0.1 s each by scanning the sample with a step size of 1 µm. This procedure was repeated for about 600 evenly spaced angular positions of the sample which rotated over 180°. Our observations confirm the presence of lateral branches surrounded by a dense collar linking tubules together and a fibrillar mesh globally located in planes perpendicular to the tubule main axis in agreement with the literature. We could also distinguish local heterogeneities in this organization: the interface between ITD and PTD exhibits fibrils oriented along the tubule main axis and there are local differences in mineral density that cannot be seen on averaged/global estimations. More strikingly, the collagen fibrils 3D organization in intertubular dentin can also be observed, a direct consequence of the very high achieved resolution.