A 3D culture model study monitoring differentiation of dental epithelial cells into ameloblast-like cells
A 3D culture model study monitoring differentiation of dental epithelial cells into ameloblast-like cells
复制标题
监测牙上皮细胞向成釉细胞样细胞分化的 3D 培养模型研究
作者：
Tadaki;M.;Anada;T.;Shiwaku;Y.;Nakamura;T.;Nakamura;M.;Kojima;M.;Arai;T.;Fukumoto;S.;and Suzuki;O.
本研究旨在探讨大鼠切牙源性牙上皮细胞系SF 2的三维（3D）培养是否促进其向成釉细胞样细胞的分化。将SF 2细胞在实验室制造的培养装置中孵育，以在存在或不存在分化补充剂的情况下形成球状体。用显微镜监测球状体的大小。通过免疫荧光染色测量釉原蛋白、成釉蛋白和釉质蛋白酶KLK-4的mRNA表达或定位来分析分化。通过Von Kossa染色和使用微力传感器的硬度测试来监测球状体的矿化。透射电镜观察细胞形态。还评估了细胞凋亡。结果表明，3D培养允许SF 2细胞在一天内形成均质球状体，其在培养2天时与二维培养相比表现出基质蛋白mRNA的显著增加。在整个孵育期间（长达8天）检测到成釉蛋白和KLK-4表达，但釉原蛋白表达显著降低，表明KLK-4酶活性增加。两种培养基制剂增加矿化，硬度和细胞凋亡，虽然分化培养基往往有更强的效果。超微结构分析表明，细胞内和细胞外的分泌颗粒的存在，这可能代表基质蛋白的分泌。总之，我们的研究结果表明，球体培养可能是一个有用的模型，用于分析成釉细胞分化。
The present study was designed to investigate whether a three dimensional (3D) culture of the rat incisor-derived dental epithelial cell line SF2 enhances its differentiation into ameloblast-like cells. SF2 cells were incubated in a laboratory-fabricated culture device to form spheroids in the presence or absence of differentiation supplements. The size of the spheroids was monitored microscopically. Differentiation was analyzed by measuring mRNA expression or the localization of amelogenin, ameloblastin, and enamel protease KLK-4 by immunofluorescence staining. The mineralization of the spheroids was monitored by Von Kossa staining and by the hardness test using a micro force sensor. The cell morphology was observed by transmission electron microscopy. Apoptosis was also assessed. The results indicated that the 3D culture allowed the SF2 cells to form homogenous spheroids within one day that exhibited a significant increase in matrix protein mRNA compared to the two dimensional culture at 2 days of culture. Ameloblastin and KLK-4 expression was detected throughout the incubation period (up to 8 days), but amelogenin expression decreased markedly, suggesting increased KLK-4 enzymatic activity. Both medium preparations increased mineralization, hardness, and apoptosis, although the differentiation medium tended to have a stronger effect. Ultrastructural analysis indicated the presence of intracellular and extracellular secretory granules, which could represent the secretion of matrix proteins. Overall our results suggest that the spheroid culture could be a useful model for analyzing ameloblast differentiation.