| Objective Adult stem cells are multipotent and can be induced experimentally to differentiate into various cell lineages. Such cells are therefore a key part of achieving the promise of tissue regeneration. The most studied stem cells are those of the hematopoietic and mesenchymal lineages. Recently, mesenchymal stem cells were demonstrated in dental tissues, including dental pulp, periodontal ligament, and dental follicle. The dental follicle is a loose connective tissue that surrounds the developing tooth. DFSCs represent distinct stem cell population and are potential candidate cells to regenerate periodontal complex. The aim of this experiment was to identify and cultivate the DFSCs in vitro, and observe the osteogenesis of DFSCs in osteogenic medium, for its application in the periodontal tissue engineering.Methods DFSCs for primary culture were obtained by culturing tissue explants from healthy subjects whose third molars were being extracted for orthodontic reasons during the phase of tooth germ development. The subjects included in this study were 12 to 16 years old and did not have a history of systemic disease. DFSCs were primarily cultured by tissue and digestion method. After limited dilution and purification, DFSCs at the three passage were used for each experiment. The cells morphology was observed under the microscope, and Immunohistochemistry staining of vimentin and cytokeratin 14 was performed. Flow cytometry analysis was performed to detect mesenchymal stem cell markers CD44, CD90, CD105 and CD146. The viability and proliferation of DFSCs were examined by a CCK-8 assay. The Osteogenesis ability of the cells was determined by osteogenesis inductionon days 0, 1, 4, 7, 14 and 21. Alkaline phosphate( ALP) and Alizarin Red S staining were performed. Real–time quantitative PCR and Weston–blot were used to detect the m RNA levels and protein expression of BMP2, OCN, ALP and Runx2.Results The primary cells were seen around the dental follicle tissue after 24 hours of culture. After 3 days of culture, cells were observed under the microscope. Long spindle shaped cells and little polygonal cells were observed. After limited dilution and purification, only the long spindle shaped cells were observed. The Immunohistochemistry staining showed that DFSCs were vimentin positive and cytokeratin 14 negative. DFSCs significantly expressed mesenchymal stem cell markers CD44, CD90, CD105 and CD146. DFSCs’ growth curve is “ S” shape. Cells grew slowly at first and showed a trend of rapid proliferation at 3 days. At 10 days, cell proliferation was stagnant. After osteogenic inductionfor 7 days, ALP staining was positive. The expression of ALP was enhanced with the increase of induction time. Microscopically, alizarin red staining showed obvious calcium nodules after osteogenic inductionfor 14 days. Calcium nodules increased with the increase of induction time. With the increase in induction time, the osteogenesis related gene of ALP, OCN, BMP2 and RUNX2 was significantly expressed( P<0.05). The protein expression of BMP2, OCN, ALP and RUNX2 was similar to the m RNA level. On day 7 of induction compared to day 4, the amount of protein expression significantly increased(P < 0.05), and then gradually became stable( P > 0.05) after 7 days.Conclusion DFSCs are easy to harvest for culture, and there are no ethical issues. They have a low immunogenic potential and stronger proliferation and osteogenesis ability. Therefore, DFSC might be a novel possible candidate cell source in periodontal tissue engineering, for the treatment of periodontal defects in periodontitis. |
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