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Study on Differentiation of Adipose Derived Stem Cells into Odontoblast-like Cells Induced by Rat Apical Bud

Author: SunYaJuan
Tutor: LiuLuChuan
School: Third Military Medical University
Course: Clinical Stomatology
Keywords: Apical bud epithelial cells Adipose-derived stem cells Odontoblast Differentiation
CLC: R78
Type: Master's thesis
Year: 2008
Downloads: 41
Quote: 0
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Abstract


The background tooth development is a dental epithelial and mesenchymal mutual induction, the complexity of the interactions of biological processes. Induction of dental epithelial odontogenic mesenchymal further differentiate into dentin and pulp, and the dental epithelial development of the formation of the enamel organ also odontogenic mesenchymal induction. So, dental epithelial morphogenesis in the teeth to play an important role in the development and maturation. Stem cells in tissue engineering, there are two main embryonic stem cells and adult stem cells. However, due to the application of embryonic stem cells by ethics, tissue engineering only from adult stem cells considerations. Adipose-derived stem cells into stem cells one having the characteristics of all stem cells, in vitro culture a number of differentiation ability, can be divided into osteoblasts, chondrocytes, fat cells, vascular endothelial cells, muscle cells, cardiac cells and neural cells . Compared with other adult stem cells adipose-derived stem cells have the drawing easy to get rate characteristics of the tissue damage, it is in tissue engineering has an important position, is a good seed cells. The aim of the present study was designed to investigate the in vitro and in vivo experiments rat incisors Apical bud epithelial cells induced adipose-derived stem cells to odontoblast differentiation. Method 1. Rats Apical bud culture and identification of epithelial cells 4 d after birth, the rat pups incisors Apical bud organizations of using enzymatic digestion and differential digestion of primary cultured rat incisors Apical bud epithelial cells and detected by immunohistochemical staining CK14, anti-amelogenin, anti-vimentin. 2 rat adipose-derived stem cell culture and differentiation capacity of 4 d after birth, the rat inguinal adipose tissue the enzymatic digestion primary cultured adipose-derived stem cells. Oil red O staining of adipogenic; Alizarin Red staining osteogenic; nerve cell molecular markers neuron-specific enolase, S100 protein immunocytochemical staining identified neural induction. 3 the rat incisor Apical bud epithelium induced adipose-derived stem cells (ADSCs) into dentin 3.1 in vitro differentiation experiments: well-grown third-generation rat adipose-derived stem cells, culture in of Apical Bud epithelial cells the conditioned medium, observed cell growth and morphological changes. 7 days after the induction, by immunohistochemistry assay the dentin sialophosphoprotein (DSPP) expression, RT-PCR to detect the expression of dentin phosphoprotein (DSPP) and dentin matrix protein (DMP-1). 3.2 In-vivo experiment: take the good growth of the third generation of the adipose-derived stem cells, adding 5% fetal calf serum DMEM/F12 culture medium to prepare a cell suspension, good Apical bud epithelium was separated into the suspension and centrifuged to obtain recombinant cell mass, adding 20% ??fetal bovine serum DMEM/F12 medium overnight culture at 37 ° C, 5% CO2 saturated humidity conditions. The next day the recombinant cell mass of rats implanted under the kidney capsule. Renal subcapsular drawn after 8 weeks, respectively, by HE staining, Masson tri-color method and immunohistochemistry for histological detection method for transplant product. 1 rat Apical bud epithelial cells: primary epithelial cells obtained by enzymatic digestion of mixed cell elongated fusiform cells of fibroblast-like cells were polygonal epithelioid cells, epithelioid cells, fibroblasts like cells surrounding the pattern was \After 2 to 3 times the difference digestion, into fibroblast-like cells is completely removed, the adherent cells are all epithelial cells. Immunohistochemical detection of CK14 expression was positive; the amelogenin expression of positive; anti-vimentin negative expression. Rat adipose-derived stem cells: primary cells were small polygonal or short spindle, monocytes, and sometimes protruding more, and there are some fatty droplets cells after passage cells decreased fatty droplets, fat stem cell processes to reduce was small polygonal or short spindle, rapid growth, the formation of the spontaneous calcium nodules, no passage is not seen to mature adipocytes. The adipogenic, oil red O staining visible red lipid droplets particles, confirmed ADSCs adipogenic differentiation. Osteogenic induction, Alizarin red staining visible red-orange color calcium deposition confirmed ADSCs osteogenic differentiation. Adipose-derived stem cells induced to differentiate into nerve direction, neuron-specific enolase, S100 protein staining were positive results confirmed ADSCs into neuronal differentiation. Induced in vitro experimental results: CONDITIONED MEDIUM the Apical Bud epithelial cells adipose-derived stem cells can be induced morphological changes are visible cell becomes long, and a polarity change, the cells were arranged in parallel trend. The immunohistochemical visible DSPP positive staining. RT-PCR analysis showed that DSPP and DMP-1 expression. 4 in vivo induction of experimental results: HE staining of the Apical Bud epithelial cells and adipose-derived stem cells recombinant renal subcapsular product visible tube-like dentin and bone-like dentin-like structure; Masson tri-color method can be observed green dentin like structure; Immunohistochemical detection of CK14 staining positive the DSPP staining positive. Conclusion enzyme digestion and differential digestion to primary cultured the Apical Bud epithelial cells of rat incisors; 2 enzyme digestion primary cultured rat adipose-derived stem cells, and confirmed that the differentiation of multipotent capacity; Apical bud epithelial cells in vitro can be induced adipose-derived stem cells to odontoblast differentiation; Apical bud epithelium restructuring adipose-derived stem cells in the body can build a dentin-like structure.

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