Exploring Angio- and Neurogenic Properties of Human Epithelial Tooth Organoids

Objectives: Epithelial cell rests of Malassez (ERM) are an enigmatic stem cell population dispersed throughout developing and established periodontal ligament (PDL), and are derived from the ameloblast-forming dental epithelial stem cells following disintegration of Hertwig’s epithelial root sheath during root formation. ERM are localized in clusters or networks throughout the PDL, are innervated and frequently associated with blood vessels, and are generally quiescent. ERM are attributed important roles regulating periodontal homeostasis and (orthodontic) tooth movement, can directly contribute to cementum and PDL following epithelial-to-mesenchymal transition, and can acquire ameloblast-like features. In this study, we aim to shed light on the paracrine signaling factors by which the ERM exert these diverse functions.
Methods: Recently, we established novel 3D in vitro models of ERM from human called epithelial tooth organoids (TO) mirroring in vivo ERM and capable of differentiation toward ameloblast-like cells. Using single-cell transcriptomics and screening by antibody array, we aim to explore the secretory profiles of human TO as avatars for in vivo ERM, and we map the presence of angiogenic and neurogenic proteins in their secretome.
Results: Single-cell transcriptomic analysis and initial antibody array screening reveal TO express pro-angiogenic factors such as CXCL5, IL6, IL8, VEGFA and uPAR. Further confirmation will be performed by enzyme-linked immunosorbent assay (ELISA) and functionally validated by endothelial cell transwell migration assay, tube formation assay, neurite outgrowth assay as well as co-culture with endothelial cells or mouse dorsal root ganglion explants. In vivo validation will be performed in situ, using immunohistochemistry and/or RNA probes.
Conclusions: Better understanding of these puzzling cells not only holds potential to unravel fundamental aspects of tooth development, tooth movement and periodontal biology, but also in advancing tissue engineering of artificial enamel organ – bringing us one step closer to biological enamel repair and regeneration.