| Nervous system falls into central nervous system and peripheral nervous system.Peripheral nervous system injury is a clinically common disease often caused byexcessive stretching, cutting, oppressing, firearms, electric burn, radiation burn andiatrogenic injury. Without timely and effective repair after injury, muscles in the areacontrolled by injured nerve will atrophy, which will result in the dysfunction of nervousmuscle and have bad effect on future recovery.Due to the unique structure and complex cell physiology of peripheral nervous,after injury it is difficult to conduct rapid and effective spontaneous recovery. Clinically,the main symptoms after nervous injury are sensory disturbance, dyskinesia anddystrophy in the nerve-controlled area. After a period of clinical treatment, there are stillsome intractable problems such as poor neurological function and neuralgia. At present,the lack of tissue engineering technology which can be applied clinically keeps therepair after peripheral nervous injury the focus and hotspot in the basic and clinicscientific research.At present stage, the clinical treatments of nervous injury are mostly nerve autograft, drug protection and the promotion of axon regeneration, which achieves nosatisfactory effect. Histological observation of the animal model with nervous injuryshows the following main reasons resulting in bad treatment effect after peripheralnerve suture：（1）the broken ends distortion after nerve anastomosis and scar ingrowth；（2）ineffective nerve regeneration, that is, subterminal motor nerve fiber grows intodistal sensory nerve fiber sheath or subterminal sensory nerve fiber grows into distalmotor nerve fiber sheath；（3）and slow nerve regeneration, neuromuscular synapsefibrosis and so on. At present, neither epineurial suture nor perineurial suture methodcan be used to avoid the above problems. How to timely and effectively recover the bestfunction of injured peripheral nerve tissue is still a big headache for medicalprofessionals.In recent years, the tissue engineering technology, as an interdisciplinary across lifescience and engineering science, has achieved great development and application. Itsbasic theory is establishing a bionic tissue engineering framework to support cellgrowth. With the gradual recovery of tissue and organ, the frame material can bereplaced by its own tissue through its degradation. The degradation products can beturned into harmless molecules and eliminated from the body. In addition, with thegradual cognition of this technology, by putting special and targeted cytokines on theframe we can effectively accelerate the regeneration and recovery of injured tissue. Atthe same time, people put forward many advanced viewpoints on the application of thistechnology to repair peripheral nerve injury. An ideal nervous repair sheath should havethe following structure and composition: a frame material should be bionic design toprovide good mechanical performance and plasticity. Meanwhile, shell of pipe hascellular structure to ensure the favorable exchange of nutrients and discharge ofmetabolite of tissue regeneration. But the pore should not be too big in order to preventthe ingrowth of fiber scar; b adding nerve nourishment factor can effectively control itsrelease; c the speed of sheath degradation should be in accordance with that ofperipheral nerve growth. Otherwise, before the nerve recovers completely, the sheathhas degraded without effect. This research makes the first attempt to construct axon guidance nerve sheath withchitosan as the main body and combining FK506and Netrin-1factor by usingelectrostatic spinning technology. To reach the above requirement, this experimentconducts the following studies:(1) establishing high flux drug screening core by meansof micro-fluidic technology, the best concentration of local use of FK506and Netrin-1nerve axon guidance factor is determined;(2) the preparation and performancemodification of chitosan compound FK506and Netrin-1nerve sheath;(3) testing thephysical and chemical properties and biosecurity of nerve sheath.Experiment1： The research of using micro-fluidic chip to screen the effectiveconcentration of local use of FK506and Netrin-1Objective: to design and make micro-fluidic chip used in the experiment andscreen the best effective concentration of local use of FK506and Netrin-1Method: based on Reynolds effect, design the micro-fluidic chip containingconcentration gradient generator and cell culture chamber. Using fluorescence imaging,verify the ability for the chip to screen the drug effect on nerve regeneration. Isolatedculture and amplify rat’s Schwann cell and dorsal root ganglion cell. After the cellinoculated into chip, use CCK-8kit to test Schwann cell proliferation level andmeanwhile measure the growth length of dorsal root ganglion cell axon. Analyze theeffective concentration by Liquid Chromatography-Mass Spectrometry. And test theresult reliability of micro-fluidic chip by transplanting drugs into96-well plates.Result: Micro-fluidic chip is successfully constructed. The reliability ofconcentration gradient of the chip formation is verified by fluorescence intensity. Usingchip obtains the best concentration of local use of FK5061.786±0.014ng/ml and thebest concentration of local use of Netrin-151.42±0.14ng/ml.Conclusion: Micro-fluidic chip can effectively screen the action concentration ofdrugs, successfully cultivate rat’s Schwann cell and dorsal root ganglion cell and screenthe best concentration of local use of FK506and Netrin-1. Experiment2： The research of using electrostatic spinning technology toconstruct PLGA/chitosan compound FK506and Netrin-1axon guidance nervesheath.Objective:1.Construct electrostatic spinning equipment with single electric fieldand double nozzle.2. Prepare FK506and Netrin-1axon guidance nerve sheath.3.Evaluate the structure and physical and chemical properties of nerve sheath.Method: Construct electrostatic spinning equipment according to experimentalrequirements. Reconstruct the equipment to reach the demand for double nozzleco-spinning. Use single electric field and double nozzle technology to conductco-spinning of the blended solution of PLGA and chitosan/FK506/Netrin-1, andconstantly adjust the ratio of each parameter to obtain compound nerve sheath. SEMobservation of the sheath. Mechanical machine test the elasticity modulus of the sheathand measure the basic physical and chemical properties of the sheath.Result: Construct and improve the electrostatic spinning equipment. Obtain theelectrostatic spinning solution ratio which meets the best concentration of local use ofFK506and Netrin-1, and prepare axon guidance nerve sheath. Nerve sheath has thespinning nano-fiber structure. The average diameter of compound sheath fiber is197±9.9nm. The porosity is76.25%. The water absorption is11.2%. The elasticitymodulus is1.525±0.116in the dry state and0.819±0.125in the wet state.10weeks afterin vitro degradation of sheath, the weight is0.138g. Degradation rate is47.26%. FK506and Netrin-1factor can provide the best effective concentration within45days.Conclusion: The constructed electrostatic spinning equipment with single electricfield and double nozzle meets the experimental requirements. Obtain the electrostaticspinning solution ratio of FK506and Netrin-1factor. Prepare PLGA/chitosancompound FK506and Netrin-1axon guidance nerve sheath. Nerve sheath featuresnanofiber structure, good hydrophilia and physical and chemical properties. Experiment3: The research of the biosecurity of PLGA/chitosan compoundFK506and Netrin-1axon guidance nerve sheath.Objective：Test and evaluate the biosecurity of PLGA/chitosan compound FK506and Netrin-1axon guidance nerve sheath.Method: Evaluate the in vitro biosecurity of sheath through the injection ofleaching liquor into the rat, subcutaneous stimulation experiment, determination of PIIhemolysis test, cell proliferation assay and AO-PI stain. At the same time, implantPKH26stained rat BMSC/sheath compound into nude rat. Use whole-body fluorescentimaging system to test the biosecurity in the in vivo degradation process of the frame,and HE stain the sheath implanted into SD rat to evaluate the thickness of fibrouscapsule around the sheath, the number of inflammatory cells and the mass loss within8weeks.Result：No rat death occurs after the injection of sheath leaching liquor, negativefor acute systemic toxicity test. PII index is0, subcutaneous stimulation experiment isnegative; hemolysis ratio of sheath leaching liquor is0.058%, in accordance with the5%limit set by the nation; a large number of survival cells and individual dead cells areseen by AO-PI and PKH26stain. The thickness of fibrous capsule and number ofinflammatory cells surrounding the sheath decreased with time, inflammatory cells andfibroblasts can not invade the lumen through the wall during the degradation period.Conclusion: PLGA/chitosan compound FK506and Netrin-1axon guidance nervesheath has good biosecurity and compatibility. Degradation rate of the sheath couldmeet the requirements of nerve regeneration. Sheath has the structural basis withreduced occurrence of adverse effects for nerve regeneration. |
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