| [Objective and background] Bone defect because of various causes is one of the most difficult clinical problems. The supply and demand contradiction between the graft material and the bone defect area is the difficulty has to be faced. Therefore, the key problem in tissue engineering bone research, is to construction TEB with good histocompatibility, no rejection, easier for angiogenesis. Mesenchymal stem cells (MSC) have the potential of multi-directional differentiation. Under different induction conditions, MSCs can differentiate to osteoblasts, chondrocytes, adipocytes, nerve cells and other mesoderm and ectoderm tissue. Stem cells have played an important role in the field of regenerative medicine, especially in repairing and replacing diseased cells. Stromal cell-derived factor-1 (SDF-1) is an obvious chemotaxis factor to stem cells. Whether SDF-1 multiple partially deproteinised biological bone (PDPBB) can promote stem cell chemotaxis and promote the healing of bone defect, there is no research to prove at present. In this study, we established luciferase-labeled mesenchymal stem cells in vitro, and compounded SDF-1 onto PDPBB. The aims of this study were to explore the relationship between mesenchymal stem cells migration and SDF-1 multiple PDPBB. Four parts were included in this study:[Methods](1) Recombinant lentiviral vectors containing luciferase gene was transfected in to mesenchymal stem cell line C3H10T1/2. Puromycin was used to filter the cells. Western-blot and immunofluorescenceimaging were used to detect the transfection efficiency. Bioluminescent imaging was used to detected the luciferase-labeled mesenchymal stem cells in vivo.(2)Manufactured the model of mice bone defects, and detected the expression of SDF-1 during different time phase by immunohistochemistry, in order to understand the process of dynamic change of SDF-1 expression after bone defect.(3) To investigate the migration of C3H10T1/2 cells in response to a gradient of SDF-1 in a Transwell chamber assay.(4) Compounded SDF-1 onto the PDPBB. To investigate the maigration of C3H10T1/2 cells and the SDF-1 compounded PDPBB in vitro and in vivo. Bioluminescent imaging was used to detected the luciferase-labeled mesenchymal stem cells in vivo. Postoperative X-ray and H-E biopsy were used to explore the relationship between mesenchymal stem cells migration and SDF-1 multiple PDPBB, and the tolerance as well.[Result](1) Western blotting analysis showed that the size of this protein in cells expressing about 60KD. Showed that infected C3H10T1/2 cells expressing luciferase protein. Immunofluorescence, compared with the uninfected control group, virus-infected cells group of about 100% of the cells were positive for red fluorescence. Show that has built a good stable expression of luciferase C3H10T1/2 cells. Luciferase-labeled mesenchymal stem cells were detected by Bioluminescent imaging in vivo successfully.(2) The expression of SDF-1 was higher in the side of bone defected than the normal side.(3) SDF-1 facilitated the migration of C3H10T1/2 cells in vitro.50ng/ml SDF-1 can facilitated the migration of C3H10T1/2 cells, while 200ng/ml SDF-1 and 400ng/ml SDF-1 showed no difference.(4)The migration of C3H10T1/2 cells was more efficiency in the group of SDF-1 multiple PDPBB than the PDPB alone.(p<0.01).C3H10T1/2-luci cells were detected by Bioluminescent imaging after surgery in the group bone defected, PDPB, SDF-1 multiple PDPBB. The result showed that SDF-1 multiple PDPBB had a chemotactic effct on C3H10T1/2-luci cells. X-ray showed that[Conclusion](1)A monoclonal cell line C3H10T1/2-luci stably and highly expressing luciferase was obtained.(2)The expression of SDF-1 was higher in the side of bone defected than normal side.(3) SDF-1 facilitated the migration of C3H10T1/2 cells in vitro.(4) The migration of C3H10T1/2-luci cells was more efficiency in SDF-1 multiple PDPBB than the PDPB alone both in vitro and vivo. |
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