Developing an in vitro 2D model for ameloblast secretion

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Supervisor:
Dr. Jobbágy Ákos Andor
Department of Measurement and Information Systems

The structure and function of our teeth are very complex, which makes their replacement difficult. A thorough understanding of tooth development can facilitate the development of tissue regeneration techniques.

The ameloblast cells are responsible for enamel formation. They differentiate during the bell phase of the process of tooth development. The genesis of the main enamel component, called hydroxyapatite, creates a huge amount of protons, causing local acidification. Without a buffering mechanism, enamel formation stops. Ameloblasts are probably not only responsible for the secretion of minerals that comprise enamel, but also for pH regulation by bicarbonate secretion. So far there is no appropriate model for functional measurements of these transport processes.

The aim of my thesis is to create a two-dimensional in vitro model that simulates in vivo ameloblast function in that cells form tight monolayers and express characteristic markers. This model is appropriate for physiological measurements with microfluoromerty and molecular biology methods.

I used a cell line of dental epihelium origin, displaying ameloblast-like characteristics, called Hat-7. I cultured them on Transwell membranes to create a polarized monolayer in normal and differentiating media. I checked the tightness of the monolayer by measuring transepithelial resistance. I characterized the model by measuring the expression of ameloblast markers and ion transporters.

The results obtained so far show that differentiating media markedly increased transepithelial resistance, demonstrating the formation of tight junctions. Cells expressed the tight junction proteins zonula occludens 1 and 2, occludin, claudin 1, 4, 7, 8 and occludin, as well as the ameloblast markers amelogenin first transcript variant and enamelin. The polarization of Hat-7 cells was demonstrated by microfluorometry. Cells could accumulate bicarbonate ions through their basolateral membrane, which is essential for successful transcelluler bicarbonate transport by ameloblasts.

All things considered, Hat-7 cells could be promising models of the pH regulation of ameloblasts.

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