| ObjectiveCranial neural crest cells belong to pluripotent stem cells that have the potential of differentiation into multiple tissue cells. They migrate from neural tubeand settle in mandibular at early stage of embryonic development, and are the important source of cells for cranial and maxillofacial organs, especially for tooth development. In recent years, it had been become one of hottest research topics in the field of teeth tissue engineering. In this experiment, we obtained ectomesenchymal stem cells in vitro from maxillofacial tissues of SD rat embryonic, then sorted them out by the flow cytometry technology to get p75NTR positive, i.e. p75+EMSCs, which represent the cranial neural crest origin. Based on the above steps, we discussed the biological characteristics and differentiation of p75+EMSCs in vitro, in order to reveal the mechanism of teeth occurrence and development, and to promote the development of tooth tissue engineering.Methods1 The histological identification in situ of p75+Cells’migration to facial processes. Maxillofacial tissue were dissected from E12.5d SD rat embryos, fixed, and decalcified. Microscopic observation after paraffin embedding, sectioning, then hematoxylin and eosin (H.E.) staining and dehydration, sealing piece conventionally. Taking the specimens of paraffin slice, closed in fetal bovine serum albumin(FBS), immunostaining were performed using the following primary antibodies:mouse anti-rat CD29, CD44, CD90, mouse anti-rat IgM Stro-1 and rabbit anti-rat p75NTR. Sections were then incubated with secondary antibodies and rinsed with PBS, prior to observation with a confocal laser scanning microscope.2 The obtaining and identification of P75+EMSCs (ectomesenchy-mal stem cells, EMSCSs) in vitro. We dissected rat embryos from E12.5 d SD rat, dissected maxillary and mandibular tissue under stereo microscopic, cutted up organization, digestion, centrifugal, abandoned supernatant, heavy suspension cells, cultured it in DMEM/F12 with 10% FBS (100 mμ g/ml penicillin streptomycin and 100 units/ml), and cultivated it in the constant temperature incubator. When the first generation of EMSCs grew to 80%, we labeled them with fluorescent p75NRT antibody. Then we collected p75+EMSCs and p75’EMSCs by flow cytometry sorting. We then identified the multi-directional differentiation potential of p75+EMSCs from cell morphology, cell cycle, FCM respectively, at the same time compared their biological characteristics with p75-EMSCs and EMSCs.3 A study on the Proliferation activity and stability of p75+EMSCs in vitro3.1 Growth curve drawing and doubling time calculation of p75+EMSCs. We collected the third and ninth generation of p75+EMSCs, cultured in 24 orifice with 1×104/holes, with 3 holes in each group. We took one group each day, and made it digested by 0.25% of trypsin in a span of 11 days, then drew the growth curve. Population doubling time was calculated with TD=t×[log2/(logNt-logNo)].3.2 Proliferation activity detection of p75+EMSCs. The third and ninth generation of p75+EMSCs were collected and cultured in 100ml culture bottle with 1×105/holes and 96 orifice with 1×103/holes respectively. Then we prepared samples, observed the proliferation activity of p75+EMSCs in vitro by cell cycle analysis and MTT test, and comparatively analyzed the change of amplification proliferation activity in vitro of p75+EMSCs.3.3 Detecting the cell cycle of p75+EMSCs. We collected the third sixth and ninth generation of p75+EMSCs, p75-EMSCs and EMSCs, digested them by 0.25% trypsin with 1% EDTA, centrifuged, fixed in 70% icy ethanol and kept them in 4℃ for 12 h. Later we recorded and analyzed the proportion of each phases in the cell cycles of the three kinds by flow cytometry(FCM).3.4 Analysis of p75+EMSCs cell phenotype. We collected the third sixth and ninth generation of p75+EMSCs、p75-EMSCs and EMSCs respectively, and analyzed the expression of p75NTR、Stro-1、CD29、CD44、 CD90、CD105、CD146、CD45 by FCM in order to test the purity, stem cell properties, stability and cell phenotype of p75+EMSCs in vitro.Statistical methods:SPSS 17.0 statistical software was applied to do the t-test of the sample data of PDT, MTT and flow cytometry cycle activity of p75+EMSCs, with a double side P<0.05 showing the difference was statistically significant.4 A Study on differentiation of p75+EMSCs in vitro. We collected the third generation of p75+EMSCs、p75-EMSCs and EMSCs, then respectively extracted total protein after cell lysis. Later we used Western Blot to detect AP-2β which was the surface marker of sneural crest origin cell,α-SMA which was the surface markers of Smooth muscle cell, Barx-1, Dlx1, Dlx5 which were tooth related genes, and CD339 and DKK1 which were cementum specific related genes. We discussed the differentiation fate and influencing factors of p75+EMSCs in vitro from the level of protein to clarify its cranial neural crest origin and analyze its relation with into teeth start control.Results1 SD pregnant rat embryonic HE staining results showed that there was a thin layer of epithelial cells at E12.5d facial process peripheral, and a large number of ectomesenchymal cells in the middle, and no epithelial invagination or dental epithelial tissue was identified. Observation under laser confocal microscope showed that pregnancy 12.5d rat embryo facial process had a strong expression of p75NTR, Stro-1, CD29, CD44, and CD90.2 The positive sorting rate of p75+EMSCs was 6.1%, and the growth state was good, with morphology was similar to fibroblast. The result of FCM identification showed that the positive rate of Stro-1 and p75+NTR on EMSCs before sorting were 30.20% and 84.49% respectively, and the positive rate of Stro-1 and p75+NTR on EMSCs after sorting were 96.90% and 96.62% respectively.3 The growth curve of p75+EMSCs was in the shape of "S", and the cell proliferation ability was strong, showing no obvious sign of stagnant period. MTT analysis showed that a slightly higher OD value was detected for the 3rd generation p75+EMSCs from day 5 to 7 compared with the ninth generation. Cell-cycle analysis showed a similar percentage of cells in the S phase, with respective numbers of 24.65%、16.21%、20.77%. The special substances that included CD29, CD44, CD90, CD105, CD146, Stro-1, p75NTR were marked for p75+EMSCs, and the expressions of the markers were all higher(>90%), which means that p75+EMSCs showed strong characteristics of mesenchymal stem cells.4 Western blotting analysis showed that the expressions of AP-2β which was the surface markers of neural crest origin cell, and Barx-1、 Dlxl、Dlx5 which were tooth related genes were higher on p75+EMSCs than on unsorted EMSCs and p75-EMSCs. At the same time, the expression of α-SMA which was the surface markers of Smooth muscle cell was lower on p75+EMSCs than on unsorted EMSCs and p75-EMSCs. The expression of CD339 and DKK1 which were cementum specific related genes showed no significant difference among p75+EMSCs and EMSCs, p75-EMSCs.ConclusionsWhen SD rats was pregnant for 12.5d, its cranial neural crest cells had migrated to facial process, but tooth development had not started yet. We could obtain EMSCs representing cranial neural crest origin, namely p75+EMSCs, in vitro. p75+EMSCs had strong proliferation activity and good biological stability, and no differentiation to the smooth muscle cells was identified. By Western blotting experiment, we witnessed an obvious enhancement on teeth starting gene of p75+EMSCs, which suggests that p75NTR might be involved in the developmental process of tooth. |
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