Multi-root Development and Hertwig's Epithelial Cells in Mouse Molars

Objective: Hertwig's epithelial root sheath (HERS) cells play multi-functions in root development. In the present study, we focused on diverse root ramification of the upper and lower molars in mice to elucidate the function and fate of HERS with the aid of histology-based 3D reconstruction of HERS architecture and µCT-based 4D visualization of root process/canal development. Materials and methods: ICR mice at 0 through 28 days of age were used. Upper and lower jaws including molars were first used for collection of µCT data (Nittetsu ELE-SCAN) and then were processed for preparation of paraffin-embedded sagittal serial sections. Immunohistochemistry using cytokeratin as a marker for epithelial cells was applied to serially cut sections (4 µm thick). The labeled HERS was demarcated by means of RGB color segmentation procedures. Computer-aided 3D and 4D reconstructions were achieved with the aid of RATOC TRI-SRF2 software. Results: Time-sequential morphogenesis of the upper and lower first molars was visualized in motion by superimposing µCT-3D constructs made at animal ages. After the completion of crown formation at day 3, the inner and outer enamel epithelia fused below the level of the crown cervical margin to produce HERS. At day 6, the dentin projections rimmed with HERS became appreciable for both upper and lower first molars, followed by cellular contact, dentin fusion and closure of the pulp chamber floor at days 10-14. The cellular fusion appeared to trigger the disintegration of HERS covering the dentin projections, resulting in a number of discrete epithelial islands attached to the root dentin surface, whereas the residual HERS seams corresponding to the fusion line remained intact during the root elongation. Conclusion: The results obtained support the theory that HERS acts as a guide for diverse root ramification and elongation accompanying harmonious root dentinogenesis under HERS induction and cementogenesis after HERS disintegration.