Sakakura Y, Fujiwara N, Nawa T
Department of Oral Anatomy, School of Dentistry, Iwate Medical University, Japan.
In Vitro Cell Dev Biol. 1989 Oct;25(10):959-64. doi: 10.1007/BF02624010.
Mandibular first molars from 17-d-old mouse embryos were cultured in vitro for 2 to 4 d by a simple, disposable, improved floatation method. This method consisted of using a 24-well multidish and a plastic culture chamber with a membrane filter. The improved floatation method, as well as our previous method, was capable of the three-dimensional development of tooth germs. Cytodifferentiation of odontoblasts and ameloblasts and formation of extracellular matrices were accelerated by the present culture system, in comparison with our previous method. All the molars cultivated by this method were very similar in morphology to in vivo. On Day 2 of culture the terminal cytodifferentiation of odontoblasts and the formation of predentin were ascertained in the bucco-lingual sections of the cultured molars. A thick layer of predentin was formed at the tip of the cusp and gradually decreased toward the cervical loop and the fissure between the buccal and lingual cusps. On Day 4 in vitro, secretory ameloblasts produced enamel matrix, and the mineralized enamel showed showed prismatic structure very similar to that in vivo. Dentin and predentin also were normal in ultrastructure. The extracellular matrices (enamel, dentine, and predentin) were formed in line with the pattern of the cusp and the formation of matrices normally started at the tip of the cusp. We conclude that the three-dimensional development of whole tooth germs in vitro may be very important for normal expression of the developmental program intrinsic to mouse embryonic molars.
采用一种简单、一次性、改良的漂浮培养法，将17日龄小鼠胚胎的下颌第一磨牙进行体外培养2至4天。该方法包括使用24孔多盘培养板和带有滤膜的塑料培养室。改良的漂浮培养法以及我们之前的方法，均能够使牙胚进行三维发育。与我们之前的方法相比，目前的培养系统加速了成牙本质细胞和成釉细胞的细胞分化以及细胞外基质的形成。用这种方法培养的所有磨牙在形态上与体内磨牙非常相似。在培养的第2天，在培养磨牙的颊舌切片中确定了成牙本质细胞的终末细胞分化和前期牙本质的形成。在牙尖顶端形成了一层厚厚的前期牙本质，并朝着颈环以及颊尖和舌尖之间的裂隙逐渐变薄。在体外培养的第4天，分泌型成釉细胞产生了釉质基质，矿化的釉质呈现出与体内非常相似的棱柱结构。牙本质和前期牙本质的超微结构也正常。细胞外基质（釉质、牙本质和前期牙本质）按照牙尖模式形成，基质的形成通常从牙尖顶端开始。我们得出结论，体外全牙胚的三维发育对于小鼠胚胎磨牙固有发育程序的正常表达可能非常重要。
