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.
