Induction Of Rat Ectomesenchymal Stem Cells Differentiation Into Odontoblast-like Cells

It has been known as "ectomesenchyme" when cranial neural crest migerates into facial process. These neural crest-derived ectomesenchymal stem cells have the plasticity to differentiate into multiple mesenchyme derivatives such as bone, cartilage, dentin, and dental pulp. After a number of cell divisions, ectomesenchymal stem cells terminally differentiate into postmitotic tall columnar odontoblast cells responsible for the synthesis of a mineralized dentin matrix. This event is highly coordinated and regulated by extracellular matrix molecules, signaling molecules, growth factors, and their receptors(such as FGF, IGF, TGF, BMP, and so on.). During the differentiation process, precise cell-cell, cell-matrix, and matrix-matrix interactions are responsible for triggering odontoblast differentiation and synthesis of the key components resulting in matrix calcification. Researchers implicated several regulatory factors and complex interactions with the epithelial-derived ameloblasts in the regulation of odontoblast differentiation.DSPP (a gene expressed to protein, DSP and DPP), is necessary for mineralized dentin formation. Although some researchers had reported that DSPP was expressed in bone at a low level, most investigators still think of it as the special expression in odontoblast.Therefore, the aim of this study was to know whether the rat ectomesenchymal stem cells could be induced to differentiation intoodontoblast-like cells in vivo and in vitro, which can lay foundation for the study of the mechanism of tooth development and pulp-dentin restoration and tissue engineering of tooth. Some growth factors such as bFGF and/or IGF-1 were added into the medium, and some experimental methods, such as immunohistochemistry, in situ hybridization, RT-PCR and so on, were used to analyze the phenotype of the induced cells and demonstrate that they were odntoblast-like cells.There were three parts in this study:1 In vitro Culture and identification of the rat undifferentiated ectomesenchymal stem cells. The rat ectomesenchymal stem cells were isolated and purified because of the difference of the tolerance to trypsin between epithelial cells and mesenchyme cell. And their morphological traits were observed under phase-contrast microscopy, and their origination was examined immunohistochemically. The results show that the cultured ectomesenchymal stem cells appear as fibroblast-like cells, and the HNK-1, VIM, NSE, S-100 are expessed in the cells, while GFAP, NF, CK and Vffl factor presented negative. Thus, the cultured cells are ectomesenchymal stem cells and may be used to ensuing study of induction.2 Induction of rat ectomesenchymal stem cells deifferentiation into odontoblast-like cells by bFGF and IGF-1.2.1 The effect of bFGF and/or IGF-1 on the characters of rat ectomesenchymal stem cells. To detect the effect of bFGF and/or IGF-1 upon the proliferative and ALPase activity of rat ectomesenchymal stem cells, MTT method and ALPase immunocytochemistry were used to examine the proliferative and ALPase activity of rat ectomesenchymal stem cells which had been treated with bFGF and/or IGF-1. The findings indicate that bFGF can increase the proliferative activity of rat ectomesenchyme cell, while bFGF and IGF-1 may promote the differentiation of rat ectomesenchymal stem cellsconcurrently, which can provide clue for the following investigation.2.2 Induction of rat ectomesenchymal stem cells differentiation into odontoblast-like cells by bFGF and IGF-1.2.2.1 To explore the differentiation mechanism of rat ectomesenchymal stem cells into odontoblast. bFGF and/or IGF-1 were added into medium to treat rat ectomesenchymal stem cells. The results show that, the cells treated with bFGF and IGF-1 grow well, cytoplasmic process elongated, and DSP is expressed in them, while only a little cells treated with IGF-1 appear single-polar cytoplasmic process and DSP-positive expression.2.2.2 Rat ectomesenchymal stem cells were cultured in three-dimension culture model b...