Abstract
In dental pulp, diverse types of cells mediate the dental pulp immunity in a highly complex and dynamic manner. Yet, 3D spatiotemporal changes of various pulpal immune cells dynamically reacting against foreign pathogens during immune response have not been well characterized. It is partly due to the technical difficulty in detailed 3D comprehensive cellular-level observation of dental pulp in whole intact tooth beyond the conventional histological analysis using thin tooth slices. In this work, we validated the optical clearing technique based on modified Murray’s clear as a valuable tool for a comprehensive cellular-level analysis of dental pulp. Utilizing the optical clearing, we successfully achieved a 3D visualization of CD11c+ dendritic cells in the dentin-pulp complex of a whole intact murine tooth. Notably, a small population of unique CD11c+ dendritic cells extending long cytoplasmic processes into the dentinal tubule while located at the dentin-pulp interface like odontoblasts were clearly visualized. 3D visualization of whole murine tooth enabled a reliable observation of these rarely existing cells with a total number less than a couple of tens in one tooth. These CD11c+ dendritic cells with processes in the dentinal tubule were significantly increased in the dental pulpitis model induced by mechanical and chemical irritation. Additionally, the 3D visualization revealed a distinct spatial 3D arrangement of pulpal CD11c+ cells in the pulp into a front-line barrier-like formation in the pulp within 12 h after the irritation. Collectively, these observations demonstrated the unique capability of optical clearing-based comprehensive 3D cellular-level visualization of the whole tooth as an efficient method to analyze 3D spatiotemporal changes of various pulpal cells in normal and pathological conditions.
Highlights
Teeth are under continuous challenges by foreign pathogens and metabolic products of oral bacteria [1,2,3,4,5]
A small population of CD11c+ cells were observed with an odontoblast-like morphology extending long cytoplasmic processes into the dentinal tubule (Figure 1c,d and Supplementary Video S1)
The dentin was identified by dentinal collagen visualized with a second harmonic generation (SHG) signal [25,26] (Figure 1d, Supplementary Video S2), and the CD11c+ cells extending long processes into the dentinal tubule were located at the dentin-pulp interface
Summary
Teeth are under continuous challenges by foreign pathogens and metabolic products of oral bacteria [1,2,3,4,5]. In experimental dental caries or pulpitis-induced teeth, pulpal dendritic cells were observed to be accumulated along the pulp-dentin border [11,12,13]. Bhingare et al (2014) reported that the dental pulp dendritic cells can migrate to regional lymph nodes for antigen presentation to T cells with increased expression of MHC class II [15]. These studies demonstrate that the highly dynamic cooperation of odontoblasts and pulpal dendritic cells orchestrate the initial innate immune responses and the subsequent adaptive immune response [5,16,17]. We established an optimized tooth optical clearing method based on modified Murray’s clear and showed a 3D cellular-level visualization of the whole tooth by using various transgenic reporter mice expressing endogenous fluorescence protein in a specific subpopulation of immune cells [22,23]
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