Contrast-enhanced nano-CT reveals dental soft tissues and cellular layers

Aim: Nano-CT technologies offer 3D imaging methods that allow high-resolution examination of bones and teeth, but soft tissue components have weak X-ray attenuation and are not easily visualised in CT images. We introduce a methodology designed to simultaneously visualise dental ultrastructure, including cellular and soft tissue components, by utilising phosphotungstic acid (PTA) as a contrast-enhancement agent. Methodology: Sound third molars were collected from healthy human adults and fixed in 4% buffered paraformaldehyde. To evaluate the impact of PTA in concentrations of 0.3%, 0.7% and 1% on dental soft and hard tissues for CT imaging, cementum and dentine-pulp sections were cut, dehydrated and stained with immersion periods of 12 hours, 24 hours, 2 days or 5 days. The samples were scanned with high-resolution nano-CT, where we examined both the cementum and pulpal regions with pixel sizes down to 0.5 µm for dental-pulp sections. Results: Dental cementum and periodontium as well as odontoblasts and predentine were made visible through PTA-staining in high-resolution three-dimensional nano-CT scans. Different segments of the tooth required different staining protocols. The thickness of the cementum could be computed over the height of the tooth once it was made visible by the PTA-enhanced contrast, and the attached soft tissue components of the interior of the tooth could be shown on the dentine-pulp interface in greater detail. Three-dimensional illustrations allowed a histology-like visualisation of the sections in all orientations with a single scan and easy sample preparation. The segmentation of the sigmoidal dentinal tubules and the surrounding dentine allowed a three-dimensional investigation and quantative of the dentine composition, such as the tubular lumen or the ratio of the tubular lumen area to the dentinal surface. Conclusion: The staining protocol made it possible to visualise hard tissues along with cellular layers and soft tissues using a laboratory-based nano-CT technique. The protocol depended on both tissue type and size. This methodology offers enhanced possibilities for the concomitant visualisation of soft and hard dental tissues.