Abstract
Background and objectives: Enamel covers the tooth crowns and is the hardest and most mineralised tissue in the body. In humans and most mammals, enamel is complex in structure, with prominent Hunter-Schreger bands (HSBs). Disturbances in enamel formation, including simplification in enamel structure and decrease in thickness, can cause dental abnormalities compromising oral and general health. In dolphins, enamel is naturally diverse in complexity, from species with HSBs, to species with radial and prismless enamel. This pilot study used lower jaw samples from common dolphin (Delphinus delphis) foetuses to study enamel development and complexity.
Methods: Oral tissues of foetuses (total length = 72-101cm) were used to evaluate the expression of the protease KLK4 and transcription factor RUNX2 which control enamel maturation and structure. Lower jaw samples were fixed, dehydrated and Micro-CT scanned for morphological and morphometric analyses. Specimens were decalcified in ethylenediaminetetraacetic acid, cleared in xylene, and embedded in paraffin wax for immunohistochemistry (IHC) analysis. Serial sections (3 μm) were deparaffinised, hydrated and heated in trisodium citrate buffer for antigen retrieval. Sections were exposed to primary antibodies for RUNX2 and KLK4, and negative controls of mouse and rabbit IgG.
Results: The average bucco-lingual and mesio-distal diameter of the developing teeth measured in Micro-CT images were 2673 μm (±SD 677) and 1981 μm (±SD 571), respectively. The enamel thickness was 137 μm (±SD 26) at the base (cervical), 160 μm (±SD 6) mid-crown, and 175 μm (±SD 32) at the tip (incisal). KLK4 was not detected in dolphin samples by IHC under the experimental conditions tested here. Positive IHC staining for RUNX2 was observed in the oral epithelium of dolphins in two samples retrieved at 95°C.
Conclusion: While promising, an immunohistochemical approach has limitations in analyses using tissues which could have undergone enzymatic, chemical, or thermal degradation such as the dolphin samples analysed here. These findings provide a foundation for future research investigating key enamel development factors, and for determining how those might influence enamel structural complexity.