Aurélien GOURRIER

We show that autofluorescence imaging can be used as a label-free modality to visualise histological features of human teeth. The autofluorescence emitted by enamel, dentin, predentin, and interglobular dentin (IGD) allows observing structural details that would otherwise require complex staining or even complementary electron microscopy or X-ray imaging. The simultaneous acquisition of autofluorescence and conventional staining fluorescence images by confocal microscope also provides important advantages to visualise occluded dentin porosity or the presence of porosity within IGD. However, autofluorescence imaging is practically limited by photobleaching. We, therefore, quantified the photobleaching extent in 3D and showed that, in confocal microscopy, the whole illuminated volume is bleached during acquisition. To mitigate this phenomenon, we analysed the photobleaching behaviour in enamel, dentin, predentin, and IGD as a function of laser power. We found that the measured decay rate cannot be modelled by a single population decay of fluorophores, highlighting the fact that autofluorescence probably arises from multiple sources. This analysis also allowed defining a laser excitation power where the photobleaching effect saturates.

According to the current hydrodynamic theory, teeth sensitivity is mediated by odontoblast cell processes which can be activated by fluid flow in the pericellular space of bulk dentin. To better understand the possible spatial extent of such phenomena, we investigated the topology and connectivity of dentinal porosity of a healthy human tooth. Using confocal fluorescence microscopy, we modeled the porosity as a spatial graph with edges representing dentinal tubules or lateral branches and nodes defining their connections. A large fraction of porosity channels in crown dentin was found to be interconnected, with 47% of nodes linked in a single component over a millimetric distance from the dentin-enamel junction (DEJ). However, significant differences in network topology were also observed. A sharp transition in connectivity from 83% to 43% occurred at 300 µm from the DEJ, which corresponds to an early stage of tooth formation. This was reflected in all graph metrics investigated, in particular the network resilience which dropped by a factor 2. To test the robustness of our observations, an in-depth analysis of potential remaining biases of the graph extraction was conducted. Most graph metrics considered were found to be within a 10% precision range from a manually annotated ground truth. However, path metrics, which characterize transport properties, proved very sensitive to network defects. Residual errors were classified in 4 topological classes related to fluorescence staining and confocal detection efficiency, instrumental resolution and image processing. Their relative importance was estimated using statistical and physical graph attack simulations in a broad experimental range. Our modeling thus provides a practical framework to estimate the interpretability of calculated graph metrics for a given experimental microscopy setup and image processing pipeline. Overall, this study shows that dentin porosity exhibits typical characteristics of a complex network and quantitatively emphasize the importance of the smallest lateral branches. Our results could be used to model fluid flow more accurately in order to better understand mechanosensing by odontoblasts in dentin.