Study On Differentiation Of Adipose Derived Stem Cells Into Odontoblast-like Cells Induced By Rat Apical Bud

BackgroudFormation of tooth is a complicated biological process involving mutual influences between odontogenous epithelium and mesenchymal tissue. Odontogenous mesenchymal tissue can differentiate into dentin and dental nucleus induced by odontogenous epithelium, and odontogenous epithelium can also develop into enamel organ induced by odontogenous mesenchymal tissue. Therefore, odontogenous epithelium is very imporntant in the developing process of tooth.Embryonic stem cell and adult stem cell are the main two kinds of stem cells using in tissue engineering. Because of the problems of ethics about embryonic stem cell, adult stem cell becomes the main seed cell in tissue engineering. Adipose derived stem cell is one kind of adult stem cell, and it has been proved that it have the ability of multipotential differentiation. Compared with other adult stem cells, ADSC has many merits, for example, it can be gained easily with high quality and low tissue injury. Thus, ADSC has important status in tissue engineering, and is one kind of good seed cell.ObjectiveTo study the effect of rat apical bud in differentiation of ADSC into odontoblast-like cells in vitro and in vivo.Methods1. Culture and identification of rat apical bud epithelium Apical bud tissue was isolated from 4-day-old rat. Enzyme digestion and difference digestion were used to gain primary apical bud epithelium culture. CK14 was detected by immunohistochemistry for further identification.2. Culture and multipotential differentiation of rat ADSCAdipose tissue was isolated from 4-day-old rat. Enzyme digestion was used to gain primary ADSC culture. Multipotential differentiations of ADSC into fat, bone and neuron were identified by Oil red O staining, alizarin red staining, enolase and S100 protein staining respectively.3. Differentiation of ADSCs into odontoblast-like cells induced by rat apical bud3.1 Experiments in vitroADSCs were cultured in the conditioned medium of apical bud epithelium. Morphologic change was observed by invert microscope. After 7 d inducing, immunohistochemistry was carried out to detect DSPP, and mRNA expressing of DSPP and DMP-1 was detected by RT-PCR.3.2 Experiments in vivoThe passage 3 ADSCs were suspensioned in DMEM/F12 containing 5% fetal serum, and mixed with prepared apical bud epithelium suspension. Reconstituted cells mass was obtained by centrifugalization, and maintained in DMEM/F12 containing 20% fetal serum over night. Next day, the reconstituted cells mass was planted under rat renal capsule. 8 w later, the product was taken out, and underwent histological detecting by HE, Masson three colors and immunohistochemidtry.Results1. rat Apical bud epitheliumPrimary cells obtained by enzyme digestion were mixed cells, containing long spindle-shaped fibrocytes and polygonal epithelioid cells. Epithelioid cells were surrounded by fibrocytes, showing island-like shape. After 2-3 times of difference digestion, fibrocytes could be abscised, and epithelioid cells were remained. Immunohistochemistry showed positive CK14.2. rat ADSCsPrimary ADSC has few ecphymas, was small polygon or short fusiform shape, grew fast. Among the ADSCs, there were no spont white calcific nodus or mature adipose cells. After multipotential differentiation inducing tests, Oil red O staining showed red lipid droplet, alizarin red staining showed jacinth calcium salts, immunohistochemistry staining of enolase and S100 protein were both positive. All these results verified that the ADSCs gained by enzyme digestion had the ability of multipotential differentiation.3. Results of experiments in vitroInduced by rat apical bud epithelium conditioned medium, the morphology of rat ADSCs changed, for example, cells became longer, showed polarity change and parallel disposed tendency. Immunohistochemistry staining showed positive DSPP. mRNA of DSPP and DMP-1 were both detected by RT-PCR.4. Results of experiments in vivoHistological detecting of the product of reconstituted cells mass of apical bud epithelium and ADSCs showed positive results. HE showed tube-like dentin and bone-like dentin; Masson three colours staining showed green dentin-like structure; immunohistochemidtry of CK14 and DSPP were both positive.Conclusions1. Enzyme digestion and difference digestion can be used to gain primary culture from 4-day-old rat.2. ADSCs gained by enzyme digestion have the ability of multipotential differentiation.3. Induced by apical bud epithelium conditioned medium, rat ADSCs can differentiate into odontoblast-like cells in vitro.4. Reconstituted cells mass of apical bud epithelium and ADSCs can construct dentin-like structure in vivo.