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

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.

TRPM7-Mediated Calcium Transport in HAT-7 Ameloblasts.

TRPM7 plays an important role in cellular Ca2+, Zn2+ and Mg2+ homeostasis. TRPM7 channels are abundantly expressed in ameloblasts and, in the absence of TRPM7, dental enamel is hypomineralized. The potential role of TRPM7 channels in Ca2+ transport during amelogenesis was investigated in the HAT-7 rat ameloblast cell line. The cells showed strong TRPM7 mRNA and protein expression. Characteristic TRPM7 transmembrane currents were observed, which increased in the absence of intracellular Mg2+ ([Mg2+]i), were reduced by elevated [Mg2+]i, and were inhibited by the TRPM7 inhibitors NS8593 and FTY720. Mibefradil evoked similar currents, which were suppressed by elevated [Mg2+]i, reducing extracellular pH stimulated transmembrane currents, which were inhibited by FTY720. Naltriben and mibefradil both evoked Ca2+ influx, which was further enhanced by the acidic intracellular conditions. The SOCE inhibitor BTP2 blocked Ca2+ entry induced by naltriben but not by mibefradil. Thus, in HAT-7 cells, TRPM7 may serves both as a potential modulator of Orai-dependent Ca2+ uptake and as an independent Ca2+ entry pathway sensitive to pH. Therefore, TRPM7 may contribute directly to transepithelial Ca2+ transport in amelogenesis.

Development of a quantitative preclinical screening model for implant osseointegration in rat tail vertebra.

OBJECTIVES: Functional tooth replacement and bone regeneration are parts of the daily practice in modern dentistry, but well-reproducible and relatively inexpensive experimental models are still missing. We aimed to develop a new small animal model to monitor osseointegration utilizing the combination of multiple evaluation protocols. MATERIAL AND METHODS: After cutting the tail between the C4 and C5 vertebrae in Wistar rats, costume made, parallel walled, non-threaded implants were placed into the center of the tail parallel with its longitudinal axis using a surgical guide. Osseointegration of the titanium implants was followed between 4 and 16 weeks after surgery applying axial extraction force, and resonance frequency analysis as functional tests, and histomorphometry and micro-CT as structural evaluations. RESULTS: In functional tests, we observed that both methods are suitable for the detection of the time-dependent increase in osseointegration, but the sensitivity of the pull-out technique (an approximately five times increase with rather low standard error) was much higher than that of the resonance frequency analysis. In structural evaluations, changes in the detected bone implant contact values measured by histomorphometry (yielding 1.5 times increase, with low variations of data) were more reliable than micro-CT based evaluations to screen the developments of contact between bone and implant. CONCLUSION: Our results provide evidence that the caudal vertebrae osseointegration model is useful for the preclinical evaluation of implant integration into the bone. CLINICAL RELEVANCE: The combination of the biomechanical and structural tests offers a well-reproducible small animal system that can be suitable for studying the integration of various implant materials and surface treatments.

Defense Mechanisms Against Acid Exposure by Dental Enamel Formation, Saliva and Pancreatic Juice Production.

The pancreas, the salivary glands and the dental enamel producing ameloblasts have marked developmental, structural and functional similarities. One of the most striking similarities is their bicarbonate-rich secretory product, serving acid neutralization. An important difference between them is that while pancreatic juice and saliva are delivered into a lumen where they can be collected and analyzed, ameloblasts produce locally precipitating hydroxyapatite which cannot be easily studied. Interestingly, the ion and protein secretion by the pancreas, the salivary glands, and maturation ameloblasts are all two-step processes, of course with significant differences too. As they all have to defend against acid exposure by producing extremely large quantities of bicarbonate, the failure of this function leads to deteriorating consequences. The aim of the present review is to describe and characterize the defense mechanisms of the pancreas, the salivary glands and enamel-producing ameloblasts against acid exposure and to compare their functional capabilities to do this by producing bicarbonate.

No Change in Bicarbonate Transport but Tight-Junction Formation Is Delayed by Fluoride in a Novel Ameloblast Model.

We have recently developed a novel in vitro model using HAT-7 rat ameloblast cells to functionally study epithelial ion transport during amelogenesis. Our present aims were to identify key transporters of bicarbonate in HAT-7 cells and also to examine the effects of fluoride exposure on vectorial bicarbonate transport, cell viability, and the development of transepithelial resistance. To obtain monolayers, the HAT-7 cells were cultured on Transwell permeable filters. We monitored transepithelial resistance (TER) as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using the fluorescent indicator BCECF. Activities of ion transporters were tested by withdrawal of various ions from the bathing medium, by using transporter specific inhibitors, and by activation of transporters with forskolin and ATP. Cell survival was estimated by alamarBlue assay. Changes in gene expression were monitored by qPCR. We identified the activity of several ion transporters, NBCe1, NHE1, NKCC1, and AE2, which are involved in intracellular pH regulation and vectorial bicarbonate and chloride transport. Bicarbonate secretion by HAT-7 cells was not affected by acute fluoride exposure over a wide range of concentrations. However, tight-junction formation was inhibited by 1 mM fluoride, a concentration which did not substantially reduce cell viability, suggesting an effect of fluoride on paracellular permeability and tight-junction formation. Cell viability was only reduced by prolonged exposure to fluoride concentrations greater than 1 mM. In conclusion, cultured HAT-7 cells are functionally polarized and are able to transport bicarbonate ions from the basolateral to the apical fluid spaces. Exposure to 1 mM fluoride has little effect on bicarbonate secretion or cell viability but delays tight-junction formation, suggesting a novel mechanism that may contribute to dental fluorosis.