Dentin Analysis with Schmörl-Staining in the NOS-3 Knockout Mouse

Objectives: The NOS-3 knockout model is widely used to study developmental changes in animals where the systemic periferal microcirculation is strongly reduced (Fleming et al., 1999). In previous studies it has already been shown that structural abnormalities could be detected at the ultrastructural level. This was reflected in dentinal tubules that were larger than those in the wild type variant (Krage et al., 2001). The aim of this study was to investigate whether Schmörl's picro-thionin staining could be used to detect the differences in dentinal structure seen in the NOS-3 knockout and its wild type variant. Methods: 10 NOS-3 knockout mice and 10 wild type mice were euthanized at 150 days and intravitally fixed by perfusion with a 4% paraformaldehyde solution. Jaws were dissected from all animals and embedded in paraffin. Sections of 5 µm were made and stained with Schmörl's picro-thionin staining. A typical feature of this staining method is that lacunae, canaliculi, odontoblasts and dentinal tubuli turn bluish purple. The Student T-test for paired groups was applied to detect statistical differences. Results: Schmörl-staining revealed that dentin forming the cusps is less dense than in other parts of the tooth. This was observed in both the knockout and the wild type. Measurements of the dentin tubuli in a zone consisting of the first 15 µm of the (pre)dentin, revealed that the average diameter was 1,32 µm in the knockout whereas 1,21 µm in the wild type(P= 0,001419). This observation may be due to the decrease in microcirculation in the NOS-3 knockout mice. It was also observed that more structural abnormalities were present in the NOS-3 knockout than in the wild type. Conclusion: Schmörl's picro-thionin staining is a useful technique to visualize dentinal structure and can easily be applied to detect differences concerning the dentinal structure between various samples.