Three-Dimensional Observation of the Furcation Area during Multi-Rooted Tooth Formation in Rat

: Epithelial projections (EP), which form as Hertwig’s epithelial root sheath (HERS) extends, play an important role in the formation of multi-rooted teeth. However, three-dimensional (3D) observations of the process in which EP form the furcation area have not yet been conducted. To investigate the formation of the furcation area by EP, we used a 3D reconstruction model of the furcation area in rats, which form subpulpal lobes in the same manner as humans, and examined the extent of cell division around the epithelium. Wistar rats between postnatal days 3-18 (PN3-18) (n=3/age) were fixed by the perfusion of 4% paraformaldehyde solution, and the maxillae were dissected out and decalcified. Tissues were embed ded in paraffin by the conventional protocol, and 5-µm-thick serial sections and some sections for immunohistochemistry using an anti-proliferating cell nuclear antigen antibody of the frontal plane were prepared. All serial sections were stained by hematoxylin and eosin, and the structures of HERS, EP, dentin projections, and subpulpal lobes were overlayed after de-lineation every 5 sections from the images obtained of the maxillary second molar for the 3D reconstruction using ITK-snap software. Reconstructed images showed that a portion of HERS in the cervical region had extended into the dental papilla on PN3 to form EP. Four EP formed and extended with age at different speeds. On PN11, EP fused with one another in the center of the dental papilla and started to form subpulpal lobes. During this process, apical elongation in the apical direction of EP was not observed. Significant cell proliferation was detected in the dental papilla around the epithelium of the cervical loop. Collectively, these results indicate that the subpulpal lobes form immediately after the fusion of EP, and HERS extend in the apical direction following the fusion of EP.