| Peripheral nerve injury is a common trauma in clinical. Currently, long segmental nerve defect has been one of the most difficult treatment problems. Although autologous peripheral nerve transplantation can promote nerve regeneration and functional recovery, there are still many limitations in autologous nerve transplantation: secondary surgery injury and deficiency of function in the donor area. In addition, the application of autologous nerve grafts is limited by many other factors such as restricted source of the donor nerve and mismatch of receptor nerve diameter. Accordingly, accumulating studies are trying to repair the nerve defect with the application of nerve tissue engineering scaffold. However, for the long segmental nerve defect, regeneration based on scaffold materials alone is usually not ideal due to the long time of nerve growth, as well as serious complications such as target muscle atrophy. Hence, a large number of studies have shown that biological microenvironment of the scaffold is the key to partial nerve regeneration. Studies have shown that the compounding of seed cells inside the nerve scaffolds can improve the microenvironment and thus effectively promote nerve regeneration. Amongstthe seed cells, Schwann cells is one of the most commonly used as the main glial cells of the peripheral nervous system. However, Schwann cell showed a decreased synthesis and secretion of neurotrophic factors after culture in vitro, as well as declined cell adhesion ability, which limit the its ability to re-build the microenvironment for nerve regeneration. Therefore, it is promising to explore a convenient and efficient method to activate Schwann cells to maintain its biological property in nerver regeneration scaffolds. Studies have shown that the electromagnetic field, as a kind of safety, economy, noninvasive and convenient to physical method, can effectively regulate the activity of cell functions. It can promote the growth of the neurons, delay muscle atrophy, and improve wound healing. In the current study, we applied pulsed electromagnetic field(2.0 m T, 50Hz) in the activation of schwann cells. We found that pulse electromagnetic field can promote the proliferation of Schwann cells and activate the cells to secrete of a variety of neurotrophic factors(BDNF, GDNF, VEGF). Furthermore, we found that the the compounding of Schwann cells to nerve scaffolds can effectively promote the rat sciatic nerve regeneration to 12 mm under pulsed electromagnetic field environment in vivo, suggesting that pulsed electromagnetic field can activate transplanted Schwann cells, and consequently promote nerve regeneration and functional recovery.Part I: Screen the optimal parameters of pulsed electromagentic field and its effect on the biological activities of SCsObjective: Screen the optimal parameters of pulsed electromagnetic field(PMF), and investigate the effects of PMF on the biological activities of SCs.Methods: Different PMF gradients(0.5 m T, 1.0 m T, 2.0 m T, 5.0 m T, 10.0 m T; 50 Hz) were applied to SCs for 4 hours. Twenty four hours after PMF exposure, the tolerance of SCs to PMF was tested by flw-cytometry assay, the morphological feature of SCs was investigated by scanning electron microscopy, the optimal intensity of PMF was chosen according to the above resluts. Then, under the optimal intensity of PMF, different exposure times(0.5, 1, 2, 3, 4, 5, 6 and 8hours) of PMF were appllied to SCs, after 24 and48 hours of PMF exposure, the CCK-8 assay was performed in order to screen the optimal exposure time of PMF.Under the optimal parameters(2.0 m T;50 Hz, 4 hours) of PMF, the cell proliferation was tested by an Ed U-labeling assay after 12 and 24 hours of PMF stimulation, and the gene expression and protein secretion of neurotrophic factors were further assayed by RT-PCR and ELISA, respectively.Results: The apoptosis ratio of SCs at 0.5 m T, 1.0 m T and 2.0 m T(50 Hz) was in a similar range to that without PMF exposure, when the PMF was incresed to 5.0 m T and 10.0 m T, the percentage of apoptotic cells was siginificantly increased. The SEM results showed that SCs exhibited a spindle feature at 0.5 m T, 1.0 m T and 2.0 m T(50 Hz), when the PMF increased to 5.0 m T, the SCs were changed into round shape, losing its characteristic morpholofy. In addition, when the PMF increased to 10.0 m T, the SCs were almost broken with damaged membranes. The CCK-8 assay indicated that 24 and 48 hours after PMF exposure, the viability of SCs under PMF for 4 hours was best, however, the CCK-8 values under 4, 5, 6, and 8 hours of PMF stimulation showed no siginificant difference.12 and 24 hours after PMF exposure, the Ed U assay indicated that the proliferation of SCs was siginificantly increased, and the gene expression and protein secretion of BDNF, GDNF, and VEGF were significantly higher than that without PMF stimulation, respectively.Conclusion: The optimal parameters of PMF were 2.0 m T; 50 Hz, and the optimal exposure time was 4 hours. Under the optimal PMF stimulatin, the SCs could be activated properly, the proliferation of SCs was enhanced and the gene expression and protein secretion of neurotrohic factors were increased.Part two: Fabrication and properties evaluation of collagen-chitoson scaffoldObjective: To fabricate a biocompatible and biodegradable Collagen-Chitosan scaffold and further evaluate its corresponding properties.Methods: The freeze-drying technique was used to fabricate nerve scaffold with collagen-chitosan(4:1). The scanning electronic microscope was introduced to study the pore sizes and interval porosity. Furthermore, the degradation rate and water absorption swelling rate was also evaluated in vitro.Results: The collagen-chitosan scaffold was successfully fabricated and displayed a honeycomb- like pattern of the microchannels in the cross section with connections. This structure was preferable for exchange of nutrients. In addition, the data also showed that the swelling ratio, biodegradability and biomechanical properties were suitable for cell transplantation.Conclusion: The collagen-chitosan nerve scaffold has good micro-structure, physical and biological properties and shows great potential to be an alternative tissue-engineered nerve.Part three: The efficacy of nerve scaffold filled with Schwann cells(SCs) in bridging a long sciatic nerve defect under a pulsed magnetic fieldObjective: To evaluate the efficacy of combined application of collagen-chitosan scaffold, SCs and pulsed magnetic field in a long sciatic nerve defect repair.Methods: The collagen-chitosan nerve scaffold loaded with SCs was used to bridge a 12 mm sciatic nerve defect. A pulsed magnetic field(2.0 m T,50Hz) was used to activate the transplanted SCs. The animals were randomized into five groups:(1) animals treated with autograft to bridge the artificial 12 mm sciatic nerve gap(Autograft group);(2) animals treated with scaffold(Scaffold group);(3) animals treated with scaffold under an pulsed magnetic field(Scaffold+PMF group);(4) animals treated with scaffold loaded with SCs(Scaffold +SCs group);(5) animals treated with scaffold loaded with SCs under an pulsed magnetic field(Scaffold+SCs+PMF group). Twelve weeks after surgery, the efficacy of axonal regeneration was examined by transmission electron microscope analysis and fluorogold retrograde labeling. The functional recovery was evaluated via sciatic function index, electrophysiology and morphological appearance of target muscle. In additon, we also examined the expression of multiple neurotrophic factors.Results: Twelve weeks after surgery, Scaffold+SCs+PMF group animals showed much more and larger regenerated axons than in Scaffold group and Scaffold +SCs group, which was comparable to that in autograft group. The retrograde labeled dorsal root ganglion neurons and spine motoneurons were significant increased in Scaffold+SCs+PMF group animals. Additionally, HE staining of gastrocnemius, sciatic function index and electrophysiology also indicated better functional recovery of the animals treated with scaffold loaded with SCs under a pulsed magnetic field. Furthermore, RT-PCR showed that the expression of BDNF, GDNF and VEGF was up-regulated in the Scaffold+SCs+ PMF group.Conclusion: Combined application of collagen-chitosan scaffold, SCs and pulsed magnetic field expects to provide a tunable regenerative microenvironment, which holds the potential to promote axonal regeneration and functional recovery. |
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