Three-Dimensional Culture of Ameloblast-Originated HAT-7 Cells for Functional Modeling of Defective Tooth Enamel Formation.
Three-Dimensional Culture of Ameloblast-Originated HAT-7 Cells for Functional Modeling of Defective Tooth Enamel Formation.
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DOI：
10.3389/fphar.2021.682654
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发表时间：
2021
影响因子：
5.6
通讯作者：
Varga G
中科院分区：
文献类型：
--
作者：
Földes A;Sang-Ngoen T;Kádár K;Rácz R;Zsembery Á;DenBesten P;Steward MC;Varga G
背景：釉质形成，即牙釉质的形成，在组织形态水平上已被很好地理解，但其潜在的分子机制尚不清楚。成釉细胞分泌釉质基质蛋白和钙离子，也调节细胞外pH，因为羟基磷灰石晶体的形成产生了大量的质子。运输和调节过程的遗传或环境损害(如氟斑牙)会导致牙釉质缺陷，如矿化不足。目的：优化成釉细胞来源的HAT-7细胞三维生长的培养条件，并检测氟暴露对HAT-7球体形成的影响。方法：将细胞分散在Matrigel细胞外基质支架内，然后在三种不同的培养液中孵育，构建HAT-7三维结构。然后在两周的时间里监测球体的形成。RT-qPCR检测细胞离子转运蛋白和紧密连接蛋白的表达。用荧光染料Fura-2和BCECF微荧光法测定细胞内钙离子和pH的变化。结果：联合应用Hepato-STIM上皮细胞分化培养液和Matrigel可诱导3DHAT-7球体的扩张和形成。这些细胞保留了其上皮细胞的形态，并继续表达成釉细胞特异性和离子转运特异性标记基因。此外，与二维HAT-7单层一样，HAT-7球体能够调节其细胞内pH，并对细胞外刺激显示细胞内钙反应。最后，我们证明HAT-7球体可以作为研究氟暴露在成釉过程中的影响的疾病模型。结论：在Matrigel细胞外基质中培养的HAT-7细胞形成三维的、多细胞的、球形的结构，这些结构保留了它们调节pH和细胞内钙信号的功能。这一新的3D模型将使我们能够更好地了解釉质发生的分子机制，不仅在健康方面，而且在釉质形成障碍方面，如氟暴露引起的障碍。
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.
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影响因子：
4
作者：
Jalali R;Lodder JC;Zandieh-Doulabi B;Micha D;Melvin JE;Catalan MA;Mansvelder HD;DenBesten P;Bronckers A
通讯作者：
Bronckers A
影响因子：
4.5
作者：
Chen, Allen Kuan-Liang;Chew, Yi Kong;Oh, Steve Kah Weng
通讯作者：
Oh, Steve Kah Weng
影响因子：
8.1
作者：
Bakhti, Mostafa;Scheibner, Katharina;Lickert, Heiko
通讯作者：
Lickert, Heiko
DOI：
10.1073/pnas.1008117107
发表时间：
2010-08-03
影响因子：
11.1
作者：
Bartosh, Thomas J.;Ylostalo, Joni H.;Prockop, Darwin J.
通讯作者：
Prockop, Darwin J.
影响因子：
3.6
作者：
Jalali, R.;Guo, J.;Zandieh-Doulabi, B.;Bervoets, T. J. M.;Paine, M. L.;Boron, W. F.;Parker, M. D.;Bijvelds, M. J. C.;Medina, J. F.;DenBesten, P. K.;Bronckers, A. L. J. J.
通讯作者：
Bronckers, A. L. J. J.