The Research Of Injured Nerve Repair And Nerve Scarring Inhibition Materials

Peripheral nerve injury is a kind of common clinical trauma,the patients' sensorimotor function are severely impacted.In recent years,nerve conduits made by bioactive materials are developed to bridge the nerve injury and appeared very good treatment effects.However,the evaluation of biological activity material inhibition mostly remains at the tissue and cell levels,the research about how the appropriate nerve conduits provide microenvironment for nerve regeneration at the molecular level of and how to promote nerve regeneration is very little known.Although surgery can bridge the nerve ends with nerve conduits,possible nerve neuroma solution strategy is rarely reported.This paper aims to explore how the neurological damage repair materials-PRGD/PDLLA nerve conduits with catheter microenvironment to enhance nerve regeneration,and what is the molecular mechanism based on the factors related to lesions for the promotion of axons regeneration,and meanwhile design nanoparticles drug delivery system PGMA/PXS64/PEI for nerve scarring inhibition.We try to develop some solutions for repair of nerve injury and neuroma problem,provide some experimental basis for neural repair engineering.PRGD/PDLLA nerve conduits were used to repair rat sciatic nerve in vivo: results showed that the rats in conduit groups showed no surgical death and infection during the experimental period,recovery of surgery legs are good.Rat regenerated nerve and gastrocnemius paraffin section were stained by HE,nerve fibers and muscle fiber diameter in PRGD/PDLLA group were greater than that in PDLLA group,slightly smaller than the normal control group.Less connective tissue ingrowth was seen,the overall recovery in good condition.To better determine nerve fiber diameter size,semithin sections were made and stained by methylene blue,the results showed that nerve fibers in PRGD/PDLLA group mainly focused on three to seven microns in diameter,27.5% more than of the PDLLA group,only 4% less than normal group(31.5%).By TEM images,PRGD/PDLLA group of nerve fiber thickness of myelin sheath is mainly focused on between 0.2 to 0.4 microns,21% more than that of the PDLLA group,only 11% less than that of normal group.The animal experiment result analysis above indicated that PRGD/PDLLA nerve conduits promoted neural axon regeneration,but changes of molecular induced by microenvironment during nerve regeneration still need to further explore.In order to answer this question,we used PRGD/PDLLA conduits to provide catheter microenvironment and determine how to promote axons regeneration at molecular level.During the nerve regeneration process,CNTF expressions in two conduits group were increased compared with normal group.CNTF expressions at two time points in PRGD/PDLLA group were greater than that in PDLLA group.Increasing CNTF stimulates the activity of the downstream signals,such as p-Akt,p-ERK and p-STAT3 in two time points.By immunohistochemical staining,positive rate in two time points in PRGD/PDLLA group were higher than that of PDLLA group,and lowest in the normal group.During the nerve regeneration process,these signal pathways are activated,and then have effects on regulating axon regeneration.Signaling pathways regulated downstream nucleus gene transcription of cytoskeleton,such as GAP-43 and tubulin during the regeneration.Expressions of GAP-43 and tubulin in PRGD/PDLLA group at two time points were higher than PDLLA group and the normal group,which means that cytoskeleton proteins were regulated to aid axon regeneration.To solve the problems after repair neuroma,we utilized PGMA as a carrier material,package PXS64 to form a drug delivery system to inhibit scar formation.PEI was used to modify the microsphere surface,make delivery system with positive charge to increase its biocompatibility.PGMA/PXS64/PEI drug-loading nanoparticles were prepared using simple method of solvent evaporation.Analysis of dynamic light scattering showed that PGMA/PXS64 microspheres had a mean size of 125 nm,surface Zeta potential measurement of 0.625 mV.After the PEI to charge modified PGMA/PXS64/PEI microsphere mean size is 154 nm,surface Zeta potential measurements is + 27 mV.We found the uniform microspheres morphology,uniform particle size distribution,and the average particle diameter of around 50 nm under TEM.The drug loading of microsphere was characterized by HPLC analysis,PXS64 drug loadings of nanometer microspheres was up to 3.06%.MTS experimental results for primary fibroblast scar cells co-cultured with PXS64,PGMA/PXS64 and PGMA/PXS64/PEI in 72 h the showed that the materials have no side effects on cell growth.We grafted the PGMA/PXS64/PEI by rhodamine B to observe the interaction between nanoparticles system with scar cells.Confocal microscope showed that the nanoparticles drug delivery system evenly distributed in cells in the body in 24 h,fluorescent is very stable,can be a very good material for cell tracking.We use Scar-in-a-Jar cell model to evaluate the inhibition function of drug system on the expression of cell type I collagen by incubation in the “collagen induced media” for 6 days.Compared with the positive control group,the collagen area per cell in PGMA/PXS64/PEI is much smaller than that without PXS64 control group,slightly larger than that in the pure PXS64 group.The nanoparticles drug delivery system we synthesized for expression of collagen inhibition for scar cells has obvious inhibitory effects.Cell type I collagen protein expression was not significantly inhibited before drug loaded in nano-spheres,good inhibition implementation of type I collagen expression in scar cells was showed after drug loaded,but the inhibition effect is not as good as that in pure drugs group in generally.The experiments above showed the preliminary implementation of drug nanoparticles for scarring inhibition.Hydrogel is the first option to repair injured central nerous system.We use the primary BMMSC to evaluate the biological toxicity of IKVAV polypeptide and grafted IKVAV hydrogel scaffold by CCK-8 and flow cytometry tests.It is concluded that the influence on cell survival is rather small,does not cause a large number of cell apoptotic and death.Through the cell cycle and proliferation cell nucleus antigen(PCNA)expression detection,we concluded that IKVAV played a certain role to promote BMMSC growth and proliferation.The results will be helpful experimental basis for the tissue engineering scaffolds research,which based on BMMSC as seed cells,with IKVAV anchor scaffold in the applications of central nerous system repair.