| Bone defects caused by trauma, infection and tumor always present a dilemma for orthopaedic surgeons. Bone defects repair included bone regeneration and vascular regeneration. At present, therapies for bone defects include autografts, allografts and artificial bone substitutes. However, all of these methods have inherent problems and limitations. Autografts are limited by a lack of adequate supply and by donor site morbidity. Allgrafts and artificial bone substitutes are not backed by adequate cellular seeds. Nowadays, the methods of tissue engineered bone have the method compounded substitutes and cellular seeds, substitutes and active factors, substitutes, cellular seeds, and active factors. Although the experiments with these methods have gained some effect, the results do not reach the ideal effect. In these experiments there are not a suitable substitute. The freezing and drying bone still seems a fitting substitute among them.Nowadays, as a commodity, immunological rejection of freezing and drying bone has seldom seen and cell-based bone tissue engineering has emerged as a promising alternative for bone repair. So in our experiment, we applied with freezing and drying bone and bone marrow mesenchymal stem cells (BMSC) as cellular seeds. In these conditions, freezing and drying bone provided fine interspace structure and good adhesive and grow conditions. At the same time, BMSC transfected vascular endothelial growth factors (VEGF) as cellular seeds provided the seeds and stimulating factors. 1. Construction and identification of VEGF eukaryotic expression vectorObjective: to construct a high effective eukaryotic expression vector of VEGF and identify its sequence for the next experiments. Methods: hVEGF165 cDNA was taken from PT7Blue/hVEGF165 plasmid and inserted into polylinker site of eukaryotic expression vector pcDNA3 to construct pcDNA3/hVEGF165. pcDNA3/hVEGF165 was identified with the sequence. Results: The pcDNA3/hVEGF165 eukaryotic expression vector was obtained and the hVEGF165 sequence is correct. Conclusion: The pcDNA3/hVEGF165 eukaryotic expression vector was constructed successfully. 2. Expression and effect of VEGF in rabbit BMSCObjective: Eukaryotic expression vector carrying pcDNA3/hVEGF165 was transfected into bone marrow mesenchymal stem cells (BMSCs). Then, to observe the change of growth and proliferation of BMSC after transfection. Methods: Rabbit bone morrow cells were cultures in Dulbecco's Modified Eagles Medium with low glucose containing 10% fetal bovine serum. Then the expression vector of pcDNA3/h VEGF165 and pcDNA3 were transfected into BMSC. After about 14 days, the BMSC was divided into three teams, one was transfected by pcDNA3/hVEGF165, one by pcDNA3, and control group. On 1, 2, 4, 7, 14, 21, 28 following days respectively, immuno-histochemical analysis was performed to examine the expression of cell plasma and samples were assayed for alkaline phosphatase histochemistry (ALP) and for mineral deposition by Von Kossa staining respectively. Results: The transfectant of pcDNA3/h VEGF165 expressed VEGF protein in cell plasma, the others didn't. ALP and depositing calcified matrix showing positive on days of 21 and 28 were not different significantly among the three groups after transfection. Conclusion: The transfectant didn't affect the BMSC growth and proliferation. Also, the transfectant of BMSC expressed VEGF protein and induced vasculargrowth.3. A study of in vivo osteogenesis of freezing and drying bone compounded BMSC transfected with VEGF.Objective: To observe in vivo osteogenesis of freezing and drying cancellous bone compounded BMSC transfected with VEGF. Methods: To transfect VEGF into culture-expanded BMSC, then compound freezing and drying cancellous bone and implant them in the muscle pouches of rabbit. Implanted substance were taken out of 8 weeks later. Slices were observed to study the osteogenesis activity of implanted substance. Results: In the group of freezing and drying cancellous bone compounded with BMSC transfected VEGF, a...
|
PDF Full Text Request