Three-Dimensional Culture of Ameloblast-Originated HAT-7 Cells for Functional Modeling of Defective Tooth Enamel Formation

Abstract

Background: Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca2+, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization.Aims: Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation.Methods: To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca2+ and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF.Results: A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis.Conclusion: In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca2+ signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure.

Bioprinting of alginate-carboxymethyl chitosan scaffolds for enamel tissue engineering in vitro

“…In our study, SEM images revealed that Alg-CMC bioink could provide a favorable environment for HAT-7 cells to retain their round-shaped round morphology and promote their attachment to the scaffolds and be guided towards mineralization and differentiation, which are essential for the enamel-like tissue formation. This is in agreement with a previous study reporting the round morphology of the HAT-7 cells when co-cultured with dental pulp stem cells in biphasic scaffolds composed of chitosan-collagen I hydrogels where their viability and proliferation were supported [4]. ALP expression, as a marker of ameloblast differentiation and mineralization, that was detected via ALP staining of bioprinted HAT-7 cells further confirmed the mineralization potential of Alg-CMC bioink.…”

“…Among other strategies, bio-fillings that can be produced by dental stem cells could offer an ideal solution to biologically regenerate/restore the affected enamel tissue [3]. HAT-7 is a dental epithelial cell line generated by rat dental stem cells that has been used to study ameloblast differentiation and enamel formation [4]. Such epithelial stem cells could be used as a base for new promising approaches that would employ the three main elements of tissue regeneration i.e.…”

Modeling ameloblast-matrix interactions using 3D cell culture

“…HAT-7 cells derived from rat molars are functional ameloblast-like cells expressing amelogenin and ameloblastin proteins ( Kawano et al, 2002 ). They are often the cell line of choice for examining functional cell polarization ( Bori et al, 2016 ), the epithelial-mesenchymal interactions during odontogenesis ( Matsumoto et al, 2011 ), and for modeling disease ( Földes et al, 2021 ). When cultured on permeable transwell filters, HAT-7 cells formed polarized epithelial layers as characterized by apical membrane permeability to CO 2 and basolateral membrane uptake of bicarbonate ( Bori et al, 2016 ).…”

“…Reliable in vitro 3D cell culture models have proven invaluable for examining the interactions between ameloblasts and the enamel extracellular matrix ( He et al, 2010 ; Földes et al, 2021 ). In vitro and cell culture systems provide for precise control over experimental conditions and the ability to isolate and examine the role of individual components in the matrix.…”

Novel trends, challenges and new perspectives for enamel repair and regeneration to treat dental defects

“…198 It has been illustrated that HAT-7 spheroids formed in Matrigel could maintain their morphology as well as pH modulation ability and expressed ameloblast markers. 247 Therefore, regeneration of the enamel tissue using scaffold-based strategies seems as a promising approach. 248 Scaffolds have been made from both synthetic and natural biomaterials in the forms of sponges, hydrogels, and meshes for regeneration of enamel or enamel-dental tissues complexes.…”