| Peripheral nerve injury(PNI) has been a common clinical problem. The repairing and regeneration of PNI has attracted more and more attention in the field of neuroscience. For small injuries, peripheral nerves can repair on their own under suitable conditions. But long gaps must be repaired with the help of nerve grafts. So researchers have been working hard to look for right artificial nerve graft to guide and promote nerve regeneration and to accelerate reconstruction of functional. As the current gold standard for treating large nerve defects, implantation of nerve autografts suffers from several drawbacks, such as lack of source, the potential differences in tissue structure and size. So researchers have been working hard to look for right artificial nerve graft to guide and promote nerve regeneration and to accelerate reconstrction of function.The ideal artificial nerve graft must have good biocompatibility and suitable mechanical properties matching the implanted tissue. A large number of studies have shown that silk fibroin has good biocompatibility, but the mechanical properties decrease a lot during the process of degumming. Therefore, in this study silk was used as raw material to prepare silk fibroin nerve guidance conduits(CSF-NGCs) with composite structure through electrospinning and braiding. The developed CSF-NGCs had good biocompatibility and mechanical properties. Biological safety of the CSF-NGCs was evaluated. In addition, CSF-NGCs were used to bridge a 10-mm long sciatic nerve defect in rats. Histological and functional assessments were performed to evaluate the influences of the CSF-NGCs on peripheral nerve regeneration. The main contents and conclusions are as follows:The inner and outer layers of CSF-NGCs were prepared by electrospinning. Effects of solution concentration, voltage and flow rate on the morphology and size of electrospun nanofiber were studied by means of scanning electron microscope(SEM). The optimal parameters were: concentration: 18%, voltage: 21 kV, flow rate: 0.2 mL/h. Self modified knitting machine was used to braid silk net. Different braid angle and density ccould be realized by changing the speed of carrier rotating and pull-up speed of conduit. Tensile properties, resistance to surgical sutures and compressive strength properties of CSF-NGCs were measured. The effect of braid angle and weave density on the mechanical properties of CSF-NGCs were analyzed. CSF-NGCs for subsequent experiments had mechanical properties as follows: tensile strength: 16.3 N, Fpull-out / 2t value: 3.5 N/mm, load of 50% deformation: 2.1 N. All properties were comparable to what has been reported. At the same time, all mechanical properties of CSF-NGCs were better than that of nerve conduits made only by electrospinning. Implant operation and in vivo test later proved good mechanical properties of CSF-NGCs. According to requirements of operation and the test results, optimal parameter of braiding was: rotating speed of carrier: 3.6 rpm, pull-up speed of CSF-NGCs: 4.5 cm/min. Wall thickness, the surface morphorlogy, porosity, hydroscopicity and permeability were measured. The results showed that CSF-NGCs had a 3D porous nanostructure with good hydroscopicity and permeability. In vitro degradation was done to analyze changes of structure, quantity, and mechanical properties of CSF-NGCs during the degradation process. Results indicated that CSF-NGCs are biodegradable in protease XIV solution.SF membrane was prepared through electrospinning. Schwann cells and Dorsal root ganglia from l-2 day old SD rats were co-cultured with the prepared film. On the 3th and 5th day, photos were taken to record the status of the co-cultures, meanwhile, fluorescent immunocyto chemistry and scanning electron microscopy(SEM) were also Performed. MTT test were performed to determine cell viability. In addition, mRNA level and release of BDNF and NGF were detected by RT-PCR and ELISA respectively. All results showed that the electrospun silk fibroin membrane had good biocompatibility with tissue and cells around, which laid the foundation for constructing and in vivo studies of nerve conduits.Genetic experiments(including Ames test, bone marrow micronucleus test, teratosperm ratio assay), acute toxicity test, subcutaneous implant test in rabbits, cytotoxicity, acute systemic toxicity experiments, intradermal test and delayed type hypersensitivity test were performed to evaluate the biological safety of CSF-NGCs. Results showed that the prepared CSF-NGCs had no genotoxicity and cytotoxicity, skin irritation and potentially allergenic. It was biocompatible and biodegradable. The results were in line with GB / T 16886 standards on biological safety evaluation of medical devices.The prepared CSF-NGCs were used to bridge a 10-mm long defect in the SD rat sciatic nerve. Autograft group and non-grafted group were done as controls. After 1 M, 3 M, 6 M, parameters recorded on the CatWalk were analyzed to evaluate the recovery of motor function. 3 M, 6 M postoperation, thermal hyperalgesia was measured. Light microscopy was used to observe morphology of target muscle. Quantitative analysis was performed using image analysis system. 6 M postoperation, electrophysiological test and immunohistochemical staining were done. Light microscopy and electron microscopy were used to observe nerve morphology, also statistical analysis was done. Results: after transplantation, over time, all functions of CSF-NGCs and autograft group recovered gradually. Animals in augograft group recovered faster than CSF-NGCs group. 3 M postoperation, many parameters of two groups differed much. However, 6 M postoperation, there were no significant difference between CSF-NGCs and autograft groups, but differences between non-grafted group and other two groups were obvious. Detailed data for 6 M:(1) For autograft group, CSF-NGCs group, non-grafted group, the gait regularity index(RI) were 85%, 79%, 29% respectively, the sciatic functional index(SFI) of rats in autograft group, CSF-NGCs group, non-grafted group were 85%, 79%, 29% respectively;(2) In the test of heat pain threshold determination, latency of normal side, autograft group, CSF-NGCs group were 7.78 s, 9.15 s, 10.23 s respectively. For non-grafted group, rats nearly didn’t move their feet, so all values were more than 20.1 s.(3) For CMAP amplitude, autograft group, CSF-NGCs group were 10.9 ± 2.1 mV and 9.8 ± 1.8 mV respectively. The average nerve conduction velocities were 28.10 ± 4.03 m/s, 35.57 ± 3.49 m/s, and 50.00 ± 3.18 m/s in the CSF-NGCs group, autograft group, and at the normal side, respectively. No CMAP was recorded at the injured side in non-grafted group.(4) Wet weight ratio was calculated to be 0.18, 0.57, and 0.64 in the nongrafted group, CSF-NGCs group, and autograft group, respectively. Cross-section area of muscle fiber in the autograft group, CSF-NGCs group, nongrafted group were 82.9%, 74.8%, 9.8% equivalent to the normal side.(5) Results of immunohistochemistry showed that axons of both CSF-NGCs group and autograft group were in disorder and diameter of the axons is a little smaller. Little nerve fibers can be seen in non-grafted group.(6) The regenerated myelinated nerve fibers in both CSF-NGCs sgroup and autograft group exhibited a compact and uniform structure with electron-dense myelin sheath and perfect basal membrane of Schwann cells, despite the thinner myelin sheaths as compared to that in normal side. Myelin sheath thickness in the normal side, autograft group and CSF-NGCs group were 1.38 ?m, 0.78 ?m, 0.65 ?m respectively, and diameter of nerve fiber were 2.31 ?m, 1.62 ?m, 1.51 ?m. In short, all results showed that 6 M after implantation, motor and sensory function in CSF-NGCs group recovered well, which was comparable to autograft group.Achievement obtained from this study would provide new idea and new method for preparing artificial nerve grafts. Relative results would provide theoretical basis for further research and application in the field of tissue engineering of the new prepared conduit. |
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