The role of cell proliferation during human primary tooth morphogenesis

Objectives: We aim to characterize proliferating cells in human teeth during the fetal period and to understand certain molecular basis of tooth morphogenesis and in particular the genesis of the permanent successional teeth. Previous literature did not quantify proliferating cells, and focused on early stages prior to formation of the successional lamina for the permanent teeth. Methods: Human fetal specimens (H22-02933) between 64-96 days gestation were used in the study. Sections were used for immunofluorescence staining with antibody to proliferating cell nuclear antigen (rabbit anti-PCNA 1:1000, Abcam #18197). Nuclei were stained with Hoechst 33452. Percentage of PCNA+ cells was counted in 4 areas of the enamel organ: the buccal and lingual outer enamel epithelium, successional lamina and stellate reticulum. One-way and 2-way ANOVA followed by Tukey’s posthoc test were used (Prism V10.5.0). Results: Primary canines (n=6) and first primary molars (n = 15) were assessed. The first signs of the successional lamina were at 73d gestation. Prior to this stage there was no significant difference in the proliferation in the buccal and lingual outer enamel epithelium. Specimens that were older than 73 days had significantly lower proliferation on the buccal side compared to younger teeth. This relatively higher proliferation on the lingual compared to the buccal side was statistically significant when all teeth were combined. Part of the higher proliferation on the lingual was due to proliferation in the successional lamina, which gives rise to the permanent dentition. There was very low proliferation in the stellate reticulum at all stages examined. Conclusions: The budding of the permanent dentition is based on the relative drop in proliferation on the buccal side, while maintaining higher proliferation in the lingual epithelium outer enamel epithelium. Insufficient proliferation on the lingual side of the enamel organ as early as 9–10 weeks may underlie permanent tooth abnormalities such as tooth agenesis or microdontia. The molecular signaling changes that accompany these proliferation differences are the next focus of this work.