| Periodontitis is a chronic infective disease, and leads to tooth losing eventually by causing the destruction of the connective tissue matrix and the resorption of alveolar bone. Currently, the treatment of periodontitis is only limited to restrain the progress of the disease, but can not reconstruct the destroyed tissues. Tissue engineering technique, as a new method, makes it possible for the destroyed periodontal tissue to regeneration.Human periodontal ligament cells(hPDLCs) have the potential to multilinear differentiation and can be used as the seed cells for the periodontal regeneration. L-ascorbic acid2-phosphate (L-AS-2-P) can promote hPDLCs to form cell sheet by increasing extracellular matrix. Compared with the traditional cell-scaffold system, cell sheet not only can retain the biological properties of cells, but also need no scaffold as carrier when transferring. Whereas the monolayer cell sheet is still not perfect, for example, it would shrink when transferring, how to make it more convenient is the main problem to be solved.Blood supply is important for any tissue engineering product, because the blood can not only provide nourish for cells, but also keep the homeostasis of the tissue. Vascular endothelial cells (ECs) are important for the angiogenesis. But adult ECs have some defect in source and immune suppression. Adipose derived stem cells(ADSCs), which are easy to acquire and have no immunogenicity and can differentiate into ECs in conditional medium, is a good source for ECs,Objective1.To construct the pellet of hPDLCs sheet and the three layers of hPDLCs sheet.2.To induce the differentiation of ADSCs into ECs in EGM-2, and to construct PDLCs-ECs-PDLCs "sandwich-like" cells sheet.Methods1. Constructing of the monolayer cell sheet:Primary hPDLCs were obtained from the premolar and the third molar. hPDLCs of three or four passage were divided into9groups with different density and cultured in different concentrations of L-AS-2-P to form monolayer cell sheet. Then the optimal condition was selected according to the principle "the shortest time and the best intensity.2. Constructing of the hPDLCs pellet and three-layer hPDLCs sheet:The cell sheet shrank for one week in37℃and was embedded in paraffin to prepare histological sections. The cell density and the amount of ECM of the polymer were observed in the microscope after HE staining. To construct three-layer hPDLCs sheet, cell were seeded in the confocal dishes to form the first layer cell sheet, and it was covered by the other two layers which was formed in6-well plates. In order to recognize the three layers, only the fist and the third layer hPDLCs were dyed with Hoechst33258, and in the other mode, only the second layer hPDLCs was dyed. Then the three-layer sheets were observed under the laser scanning confocal microscope.3. The differentiation of ADSCs into ECs:ADSCs were obtained from subcutaneous adipose tissue after digested by collagenase. To characterize its multilinear differentiation property. ADSCs were induced into osteoblast and lipoblast in conditional medium. Then we use EGM-2to induce the ADSCs to differentiate into ECs, and the expression of CDS31was detected on day7and14.Resultsl.The pellet of cell sheet had more tenacity and was more easy to mould. Higher cell density and large amount of ECM could be observed in the microscope.2. The density and brightness of fluorescence cells were consistent with the construction mode of the three-layer cell sheet.3.ADSCs could differentiate into osteoblasts and lipoblasts, but when it were cultured in EGM-2for14days, the expression of CD31could not be detected.Conclusions1. We successfully constructed the pellet and the three-layer cell sheet of PDLCs. We could not detect the CD31during the differentiation of ADSCs to ECs, and it needs further study. |
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