The Combination Of Decellularized Extracellular Matrix With Grooved Nerve Conduit For Peripheral Nerve Repair

Peripheral nerve defect is usually caused by trauma,which a serious disabling disease.Tissue engineered nerve conduit is one of the most promising direction for peripheral nerve defect repair.Objectives:Through the combination of PLGA grooved conduit with decellularized extracellular matrix to create a new tissue engineered nerve conduit.Material characterizations,cell culture and SD rat model experiments were applied to comprehensively explore the effect of novel artificial nerve conduit and compare with autologous nerve transplantation.Methods:1)PLGA smooth nerve conduit and grooved nerve conduit were prepared by dry-jet wet spinning method.The microstructure was observed through scanning electron microscope(SEM),and the mechanical properties were analyzed using tension test and the in vitro degradation rate was evaluated.The nerves,muscles,and kidneys of SD rats were decellularized,and HE staining and DNA content test were used to confirm the effectiveness and feasibility of the decellularization method.SEM was used to observe the microstructure of the decellularized material,and the porosity of all three groups were evaluated.2)Various decellularized materials were frozen cut to thin slices with a thickness of 50 μm.Smooth and grooved nerve conduits were cut from the middle and used polylysine to coat the conduits on cover slip.The sliced decellularized materials were attached on the smooth and grooved conduit.PC 12 cells were cultured on all samples to mimic nerve cell growth.The growth of PC 12 cells was evaluated by live-dead staining,CCK-8,phalloidin and DAPI staining.The preference3)After random grouping,the right sciatic nerve defect model of SD rats was established.The autologous nerve transplantation group,the grooved conduit group filled with decellularized nerve tissue,the decellularized nerve only group,the grooved conduit filled with decellularized kidney,decellularized kidney only group and grooved conduit only group were created to repair the right sciatic nerve defect,and the left side was used as the blank control.At 16 weeks after the operation,the plantar thermal stimulation latency measurement,electrophysiological test,gait analysis,gastrocnemius test,and sciatic nerve HE,immunofluorescence and histochemical staining of sciatic nerve,transmission electron,Western Blot(WB),microscope observation of sciatic nerve,and statistical analysis of all results were performed.Results:1)The nerve conduit prepared by dry-jet spinning has microporous structures with a porosity of about 80%;the tension test verified that the conduit prepared in this experiment has good mechanical properties;HE staining and DNA determination of nerves,muscles and kidneys decellularized tissues indicated that the decellularization procedures used in this project can effectively remove cells.2)Live and dead staining showed that most of the PC 12 cells were alive on the smooth conduit,and only a few dead cells on the grooved conduit;CCK-8 results showed that the number of cells cultured on the cover slip was the highest compared with smooth and grooved conduit at 7 d,but the significant difference was only found compared with the grooved conduit.The cell number on smooth conduit was higher than grooved conduit,but no significant difference was found.Phalloidin and DAPI staining showed that the PC 12 axon length had no significant difference between all groups.The PC 12 cell number was higher on decellularized kidney compare with other tissues.3)In animal study,the results of gait analysis showed that the repairing effect of the decellularized nerve-filled grooved conduit on the motor function after nerve repair was better than that of the decellularized nerve only and the grooved conduit only.The nerve repair effect of the decellularized nerve was significantly better than that of the decellularized kidney;the latent period of thermal stimulation of the autologous nerve group was significantly lower than that of the other groups.The thermal stimulation latency of the groove conduit with nerve tissue was lower than that of the nerve group only and the groove group only,while the heat stimulation latency of the groove conduit with kidney tissue had no significant difference compared with that of the kidney and groove group only;from the electrophysiological results,the nerve repair effect of grooved conduit combined with decellularized nerve is better than that of decellularized nerve and grooved conduit only,and the nerve repair effect of decellularized nerve is significantly better than that of decellularized kidney tissue,but no groups had repair effect good as autologous nerve transplantation;WB results showed that the content of nerve repairing-related protein CALR and ANXA1 in the combination of decellularized nerve and decellularized nerve was higher than that in the decellularized nerve and decellularized nerve alone,and the content of nerve repairing-related protein CALR and ANXA1 in the decellularized nerve group was higher than that in the decellularized kidney tissue,but both of them were lower than that in the autologous nerve transplantation;gastrocnemius muscle weighing and Masson staining indicated that after grooved conduit combined with acellular nerve and groove combined with acellular kidney to repair the sciatic nerve,the degree of gastrocnemius atrophy is significantly larger than that of pure acellular nerve and pure acellular kidney;from the results of HE staining of the sciatic nerve,the cross-sectional area of the new-born nerves repaired by the grooved conduit combined with decellularized nerve group is larger than that of the grooved conduit combined with decellularized kidney group,decellularized nerve group alone,and a grooved conduit only.The grooved conduit combined with decellularized kidney group is higher than the simple acellular kidney group;the results of NF200 immunohistochemistry and S100 and NF-H fluorescence staining show that the grooved conduit can promote the repair of nerves with decellularized nerve and kidney.Overall,the decellularized nerve material is better than the decellularized kidney material.Conclusion:1)The PLGA nerve conduit prepared by dry-jet wet spinning has good mechanical properties and can be effectively combined with decellularized materials for animal study.2)Decellularized nerves have certain advantages in promoting the growth of PC 12 axons,while tha decellularized kidneys can better promote the PC 12 cell growth.3)The repair effect of grooved conduit combined with decellularized materials is generally better than pure decellularized materials and grooved conduits only.It can effectively repair rat sciatic nerve defects and could be a novel idea for repairing peripheral nerve defects.