Expression and localization of CD63 in the intracellular vesicles of odontoblasts

We hypothesized that odontoblasts release exosomes as well as dental pulp cells and focused on the exosome membrane marker CD63. Odontoblasts are well-differentiated mesenchymal cells that produce dentin. Dental pulp, a tissue complex formed with odontoblasts, releases exosomes to epithelial cells and stimulates their differentiation to ameloblasts. However, the localization of CD63 in differentiated odontoblasts is poorly understood. Therefore, herein, we aimed to reveal the expression of CD63 in odontoblasts during tooth development. We first investigated the localization of CD63 in mouse incisors and molars using immunofluorescence. In adult mouse incisors, the anti-CD63 antibody was positive in mature odontoblasts and dental pulp cells but not in pre-odontoblasts along the ameloblasts in the apical bud. Additionally, the anti-CD63 antibody was observed as a vesicular shape in the apical area of odontoblast cytosol and inside Tomes' fibers. The anti-CD63 antibody-positive vesicles were also observed using immunoelectron microscopy. Moreover, during mouse mandibular molar tooth morphogenesis (E16 to postnatal 6 weeks), labeling of anti-CD63 antibody was positive in the odontoblasts at E18. In contrast, the anti-CD63 antibody was positive in the dental pulp after postnatal day 10. Furthermore, anti-CD63 antibody was merged with the multivesicular body marker Rab7 in dental pulp tissues but not with the lysosome marker Lamp1. Finally, we determined the effect of a ceramide-generation inhibitor GW4869 on the mouse organ culture of tooth germ in vitro. After 28 days of GW4869 treatment, both CD63 and Rab7 were negative in Tomes' fibers, but were positive in control odontoblasts. These results suggest that CD63-positive vesicular organelles are important for mouse tooth morphogenesis.