Abstract
Dental pulp is composed of nerves, blood vessels, and various types of cells and surrounded by a thick and hard enamel-dentin matrix. Due to its importance in the maintenance of tooth vitality, there have been intensive efforts to analyze the complex cellular-level organization of the dental pulp in teeth. Although conventional histologic analysis has provided microscopic images of the dental pulp, 3-dimensional (3D) cellular-level visualization of the whole dental pulp in an intact tooth has remained a technically challenging task. This is mainly due to the inevitable disruption and loss of microscopic structural features during the process of mechanical sectioning required for the preparation of the tooth sample for histological observation. To accomplish 3D microscopic observation of thick intact tissue, various optical clearing techniques have been developed mostly for soft tissue, and their application for hard tissues such as bone and teeth has only recently started to be investigated. In this work, we established a simple and rapid optical clearing technique for intact mouse teeth without the time-consuming process of decalcification. We achieved 3D cellular-level visualization of the microvasculature and various immune cell distributions in the whole dental pulp of mouse teeth under normal and pathologic conditions. This technique could be used to enable diverse research methods on tooth development and regeneration by providing 3D visualization of various pulpal cells in intact mouse teeth.
Highlights
Teeth are highly structured tissues comprised of enamel, dentin, and pulp tissues
Pulpal cells include dental pulp stem cells (DPSCs), odontoblasts, fibroblasts, endothelial cells surrounded by pericytes forming vascular networks, and various immune cells including macrophages, granulocytes, mast cells, and plasma cells.[1,2,3]
Odontoblasts, which are differentiated from DPSCs and located at the pulp-dentin interface, perform the specialized function of dentin maintenance by controlling the mineralization of reactive dentin and represent the first barrier against pathogens.[4,5]
Summary
Teeth are highly structured tissues comprised of enamel, dentin, and pulp tissues. A mineralized hard tissue, the enamel-dentin matrix, surrounds the dental pulp, which is a soft connective tissue that includes blood vessels, nerves, and interstitial fluid with various pulpal cells. The 3D complicated 3D cellular-level structure of thick tissue samples rendered image reconstructed with the Z-stack imaging data without the sectioning procedure.[20] Tissue optical clearing showed a dense network of capillaries in the dental pulp, as techniques have been used for various soft tissues, such as the shown in Fig. 1d and Supplementary Video 2. Required to see the hard tissue, and as mentioned earlier, this approach is complex and time-consuming It can 3D visualization of the immune cell distribution in an optically disrupt tissue integrity and more importantly, hamper conven- cleared tooth tional methods to analyze tooth mineralization at the microscopic level. We established a modified Murray’s clear method expressing immune cells residing in the dental pulp of the intact molars was successfully visualized, as shown in Fig. 2a–d and Supplementary Videos 4 and 5.
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