Experimental Studies On Safeness Of Activated Schwann Cells In Clinical Application

Introduction:Tissue engineering supplies a new approach in repairing peripheral nerve defects. The absorbable artificial nerve conduits filled with Schwann cells could be an alternative substitute of peripheral nerve autograft. Activated Schwann cells, which are acquired through stimulating Schwann cells with "activation liquid" (patent in applying), have higher reproductive activity and secrete more growth factors. Furthermore, activated Schwann cells appear to facilitate much more peripheral nerve regeneration. An important prerequisite of activated Schwann cells utilizing in clinical application is absolute safeness, that is, no carcinogenicity and no teratogenicity. The aim of this study was to detect the malignant potential of activated Schwann cells in vitro and in vivo. This study includes four parts:Objective:We aim to find some clinically applicable techniques in adult Schwann cell culture through comparative study.Methods:Fourty adult rats were randomly divided into 4 groups. Schwann cells were harvested from bilateral sciatic nerves. Group A:Predegeneration in vivo+Fast enzyme digestion; Group B:Predegeneration in vivo+Slow enzyme digestion; Group C:Predegeneration in vitro+Fast enzyme digestion; Group:Pregegeneration in vitro +Slow enzyme digestion. Cells were passaged with cold jet or trypsinization. Comparative studies include:(1) The difference of predegeneration between in vitro and in vivo in increasing the number of Schwann cells within peripheral nerves; (2) The efficiency of harvesting Schwann cells between fast enzyme digestion and slow enzyme digestion; (3) The efficiency of cold jet and trypsinization in passage. Through comparative studies, the most suitable methods were found to culture Schwann cells in clinical application.Results:Predegeneration, both in vivo and in vitro were effective in increasing the number of Schwann cells within peripheral nerves. The number of Schwann cells expanded in vitro for 1 week, were similar with either fast enzyme digestion or slow enzyme digestion. No statistical significance was found among the four groups. Both cold jet and trypsinization were effective in passage. The differences were that cold jet could purify Schwann cells during passage, but needs skillful operators and losses a certain number of cells; and trypsinization does the opposite. Conclusion:Predegeneration in vitro alleviates patients'suffering, and more applicable in clinical medicine. Both fast and slow enzyme digestion could be use in harvesting Schwann cells from peripheral nerves. Cold jet and trypsinization should be combined in passaging Schwann cells.Objective:We aim to assess the malignant potential of activated Schwann cells in vitro through comparison with both benign and malignant cells.Methods:After dissociation from adult rat sciatic nerves, activated Schwann cells were cultured in vitro. We detected the biological characteristics of activated Schwann cells in vitro through a serial of experiments, such as successive cell morphology and growth pattern observations, immunological marker identifications, proliferation index and purity detections, wound healing assay and Transwell invasion assay, karyotype analysis and detections of gene expression profiling chips and cytokine antibody chips. And the characteristics of activated Schwann cells were compared with normal Schwann cells and malignant cells.Results:Morphology of activated Schwann cells was the same with normal ones. Proliferation activity was increased, and density inhibition was decreased in activated Schwann cells. Activation and successive passages did not influence the expression of immunological markers of Schwann cells. Proliferation rate of activated Schwann cells was higher than normal ones. After activation, the abilities of migration and invasion of Schwann cells were a little enhanced, however, much lower than malignant cells. The karyotype of activated Schwann cells was normal. The expressions of genes and proteins were under dynamic balance, and the highest viability was in the second and third passages. Conclusion:Activated Schwann cells, which were dissociated from rat peripheral nerves and expanded in vitro, have no biological characteristics of malignant cells.Objective:This study was to detect whether activated Schwann cells have carcinogenicity in vivo or not.Methods:Sufficient activated Schwann cells were harvested from rat bilateral sciatic nerves. Then the cells were were autotransplanted into axillary fossa to see whether they could produce tumors in vivo or not. Normal Schwann cells were used as a negative control, and malignant rat C6 glioma cells were used as a positive control. Fifty rats were randomly divided into 3 groups. Group A:A total of 1×107 activated Schwann cells were autotransplanted into right axillary fossa of 20 rats; Group B:A total of 1×107normal Schwann cells were autotransplanted into right axillary fossa of 20 rats; Group C:A total of 2×106 rat glioma cells were transplanted into right axillary fossa of 10 rats. Equivalent rats were sacrificed at 0.5 months,1 months,2 months,4 months and 6 months after cell transplantation, respectively. The complete gross necropsies of adjacent organs were done to find any potential hypercellular area, which were further examined histologically. The origin of cell mass was identified through immunohistochemistry. Karyokinesis was observed in sections from all cell masses after H&E staining.Results:All the 10 rats receiving C6 cell transplantation developed tumors during 0.5 to 1 month. And all rats which were autotransplanted with Schwann cells, whether activated or normal Schwann cells, were found no tumor formation. And Schwann cells could survive at axillary fossa for at least 2 months and express immunological markers.Conclusion:Activated Schwann cells could survive and express immunological markers at least 2 months in abnormal micro-enviroment in vivo, and produce no tumors. Objective:This study was to detect whether autotransplantation of activated Schwann cells have influences on descendants.Methods:Sufficient activated Schwann cells were harvested from sciatic nerves of adult female rats. Adult female rats simply resected sciatic nerves were used as controls. After autotransplantation, all rats were caged with male rats. Items of comparative detection include:pregnancy rate, the amount of foeti, body weight, body length, tail length and chromosome number. It was also observed that growth, fundamental behavioral capacity and cognition of injury till four weeks.Result:The pregnancy rates of adult female rats which were resected of unilateral sciatic nerve were 40% in 3 months. No difference was seen between autotransplantation and control group. Similarly, no significant difference was seen in parameters of newly born rats, such as live feoti, dead feoti, body weight, body length, tail length, et al. No abnormalities were found in growth, fundamental behavioral capacity and cognition of injury of newly born rats four weeks later. The chromosome number of newly born rats was 42.Conclusion:Autotransplation of activated Schwann cells did not influence the biological characteristics of descendants of adult female rats.