3D-Imaging of Whole Neuronal and Vascular Networks of the Human Dental Pulp via CLARITY and Light Sheet Microscopy

Abstract

Direct visualization of the spatial relationships of the dental pulp tissue at the whole-organ has remained challenging. CLARITY (Clear Lipid-exchanged Acrylamide Tissue hYdrogel) is a tissue clearing method that has enabled successful 3-dimensional (3D) imaging of intact tissues with high-resolution and preserved anatomic structures. We used CLARITY to study the whole human dental pulp with emphasis on the neurovascular components. Dental pulps from sound teeth were CLARITY-cleared, immunostained for PGP9.5 and CD31, as markers for peripheral neurons and blood vessels, respectively, and imaged with light sheet microscopy. Visualization of the whole dental pulp innervation and vasculature was achieved. Innervation comprised 40% of the dental pulp volume and the vasculature another 40%. Marked innervation morphological differences between uni- and multiradicular teeth were found, also distinct neurovascular interplays. Quantification of the neural and vascular structures distribution, diameter and area showed that blood vessels in the capillary size range was twice as high as that of nerve fibers. In conclusion whole CLARITY-cleared dental pulp samples revealed 3D-morphological neurovascular interactions that could not be visualized with standard microscopy. This represents an outstanding tool to study the molecular and structural intricacies of whole dental tissues in the context of disease and treatment methods.

Engineering pericyte-supported microvascular capillaries in cell-laden hydrogels using stem cells from the bone marrow, dental pulp and dental apical papilla

“…SCAPs are isolated from the root of a developing immature teeth, while the DPSCs are extracted from the inner tooth pulp of adult molars 28,58 . Further, it is well known that vasculature density differs in the root (apical area) and crown parts of the pulp 59 . Therefore, this might be the reason for the varied levels of marker expression among the stem cells.…”

Tissue clearing technique: Recent progress and biomedical applications

“…Ueda's group applied CUBIC-based cancer (CUBICcancer) analysis to investigate cancer metastasis within 13 mouse models using nine cancer cell lines ( Figure 8A). CUBIC-cancer analy- (Greenbaum et al, 2017;Jing et al, 2018;Franca et al, 2019;Hong et al, 2019) nanoparticles to micrometastases could offer a way to stop tumor growth at early phases. Kingston et al combined for patients based on the specific physiology of micrometastases (Kingston et al, 2019).…”

“…(d) Tridimensional innervation of a human premolar (left) and high‐magnification images (right) of distinctive nerve splitting patterns in coronal and furcation direction. Images adapted from Refs (Greenbaum et al ., 2017; Jing et al ., 2018; Franca et al ., 2019; Hong et al ., 2019)…”

Therapeutic Functions of Stem Cells from Oral Cavity: An Update

“…The dental pulp is a loose connective tissue located in the innermost center part of the teeth. It consists of tiny blood vessels, nerves, and various cells including dental pulp stem cells (DPSCs) [15]. DPSCs are the first discovered MSCs among oral cavity-derived stem cells; in 2000, Gronthos et al isolated the stem cell-like cell population from the dental pulp of the human third molars, known as "wisdom teeth" [16].…”