| Peripheral nerve injury is one of the common clinical diseases in public health.It not only causes serious health problems such as motor and sensory dysfunction,muscle paralysis and even lifelong disability,but also the high cost of treatment and rehabilitation brings huge financial burden to the medical system.Therefore,the repair and regeneration of peripheral nerve injury is of great significance for patients and social medical system and is the focus of attention in the field of tissue engineering.At present,for short-distance peripheral nerve defects,end-to-end suture is usually used for nerve repair,but this method is difficult to repair long-distance peripheral nerve defects.Autologous nerve transplantation is the gold standard for clinical repair of long-distance peripheral nerve defects,but there are some problems in autologous nerve transplantation,such as the shortage of donor nerve,the formation of postoperative neuroma and the matching between donor nerve and injured nerve.The development of tissue engineering technology and new materials provides a new choice for the repair of peripheral nerve injury.An ideal nerve scaffold needs to provide a suitable microenvironment for the proliferation and differentiation of nerve cells and provide sufficient mechanical support for the growth of regenerated axons.At the same time,it needs to be degraded safely in time after nerve regeneration to provide space for the growth of newborn nerves,and the degraded products can be absorbed by the body,so as to avoid nerve compression and the metabolism and safety of degraded products.At present,degradable nerve scaffolds cannot perfectly repair long-distance peripheral nerve injury because their degradation speed cannot match with nerve regeneration.Therefore,it is urgent to develop new nerve scaffolds with high biodegradation rate and nerve regeneration,so as to provide new ideas for the repair of long-distance peripheral injury.This study focuses on the complex challenge of morphological structure reconstruction and functional recovery of long-distance peripheral nerve injury,based on natural silk fibroin,polylysine and electrospinning technology,starting from the natural structure of bionic peripheral nerve and the characteristics of ideal nerve scaffold.This study was evaluated in vitro and in vivo,the purpose is to invent a new ideal nerve scaffold with high matching between biodegradation rate and nerve regeneration,so as to provide new methods and ideas for the clinical treatment of peripheral nerve injury.The main research contents and conclusions of this topic are as follows:1.Fabrication and properties of silk fibroin/polylysine nanofiber membraneRandom and aligned silk fibroin nanofiber membranes were prepared by electrospinning technology,and modified by different concentrations of polylysine to obtain random and aligned silk fibroin/polylysine nanofiber membranes.The physical and chemical properties of the prepared random and aligned silk fibroin/polylysine nanofiber films were characterized by scanning electron microscope,Fourier infrared spectrometer,optical contact angle tester,X-ray diffractometer,micro controlled electronic universal testing machine,X-ray photoelectron spectroscopy and optical contact angle tester.The performance differences of random and aligned silk fibroin/polylysine nanofiber membranes with different concentrations were compared and analyzed.The antibacterial properties and biocompatibility of silk fibroin/polylysine nanofiber membrane were evaluated by antibacterial experiment and cell experiment,and the concentration of silk fibroin/polylysine nanofiber membrane was optimized.The results showed that 0.1% polylysine modified silk fibroin nanofiber membrane had higher O-C=O conversion(90.5%),ultimate strain,hydrophilicity,antibacterial and biocompatibility.Compared with random silk fibroin/polylysine nanofiber membrane,aligned silk fibroin/polylysine nanofiber membrane showed higher fiber orientation and ultimate stress,and promoted the proliferation and aligned growth of Schwann cells.Through comprehensive evaluation,the feasibility of preparing nerve scaffold with 0.1%polylysine modified silk fibroin nanofiber membrane was clarified,which laid a foundation for the subsequent preparation of nerve scaffold and its application in nerve regeneration.2.Construction and properties of silk fibroin/polylysine biomimetic nerve scaffoldIn order to improve the mechanical properties of silk fibroin/polylysine nanofiber membrane and promote the growth of nerve cells,aligned + random + aligned three-layer silk fibroin composite nanofiber membrane was prepared by electrospinning.To simulate the nerve bundle structure of natural peripheral nerve,a three-layer silk fibroin composite nanofiber membrane was prepared into a multichannel structure.Silk fibroin /polylysine biomimetic(multichannel)nerve scaffold was formed after modification with 0.1%polylysine.At the same time,silk fibroin hollow nerve catheter and silk fibroin multichannel nerve catheter were used as the control group.The appearance and mechanical properties were analyzed by scanning electron microscope and micro controlled electronic universal testing machine.The degradability of the prepared nerve catheter was evaluated by in vitro degradation experiment.The expression ability of nerve cells on the nerve catheter for specific proteins was evaluated by immunofluorescence staining experiment.The results showed that the length of silk fibroin/polylysine biomimetic nerve scaffold was 12 mm,the diameter was 1.5 mm and the diameter of a single microchannel was about 0.3 mm.The surface fibers had a high degree of orientation.Compared with natural sciatic nerve,silk fibroin/polylysine bionic nerve scaffold had higher ultimate stress(13.19 MPa).The degradation rate of silk fibroin/polylysine biomimetic nerve scaffold was 60.82 ± 2.11% after 13 weeks,while the degradation rate of silk fibroin hollow nerve catheter and silk fibroin multichannel nerve catheter was no more than 35%.Silk fibroin/polylysine biomimetic nerve scaffold could better promote the expression of S100 and GFAP proteins.3.Application of silk fibroin/polylysine biomimetic nerve scaffold in peripheral nerve regenerationThe effect of silk fibroin/polylysine biomimetic nerve scaffold on repairing longdistance peripheral nerve injury was evaluated by establishing a 10 mm sciatic nerve defect model in SD rats.The autologous transplantation group was the positive control group.At 4 and 12 weeks after operation,the recovery of sensory and motor function was evaluated by thermal stimulation test,footprint collection,sciatic nerve function index,gastrocnemius wet weight ratio and Masson staining.At 4 and 12 weeks after operation,the degradation rate of nerve scaffold in vivo and the matching of nerve regeneration were evaluated.The nerve regeneration ability in different groups was analyzed by HE staining,TB staining,LFB staining,immunohistochemistry,immunofluorescence staining and transmission electron microscope.The results showed that compared with silk fibroin hollow nerve guide conduit and silk fibroin multichannel nerve guide conduit,silk fibroin/polylysine biomimetic nerve scaffold can promote the regeneration of injured nerve and the recovery of sensory and motor function,which was comparable to that of autologous nerve transplantation.Silk fibroin/polylysine biomimetic nerve scaffold was completely degraded within 4 weeks,repaired 10 mm nerve defect without scar formation,and it has high matching between degradation rate and nerve regeneration.At 12 weeks after operation,the effect of silk fibroin/polylysine biomimetic nerve scaffold group on promoting nerve regeneration was similar to that of autologous nerve transplantation.In conclusion,based on the repair of long-distance peripheral nerve injury,this study simulated the nerve bundle structure of natural nerve,Silk fibroin/polylysine biomimetic nerve scaffolds with aligned topology were prepared.In vitro and in vivo experiments were conducted to evaluate the matching between the degradation rate of the nerve scaffold and nerve regeneration,as well as its good role in the morphological structure reconstruction and functional recovery of long-distance peripheral nerve injury.The silk fibroin/polylysine biomimetic nerve scaffold prepared in this study provided a new method and idea for the repair of peripheral nerve injury. |
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