Conductive Polymer-based Functionalized Neural Tissue Grafts For Peripheral Nerve Defects

Background:Bridging critical-sized defects in peripheral nerves to achieve functional recovery is a clinical challenge.Inadequate regeneration of peripheral nerve axons often results in long-term partial or total sensory and/or motor impairment.Currently,the best treatment available for long-gap peripheral nerve regeneration is transplantation,however,the success of this approach is requirement for donor nerves.The nerve guide conduit?NGCs?serves as an alternative to autograft,as it connects the proximal and distal ends of nerve defects and provides physical and biochemical guidance for axon regeneration.Functionalized conductive polymers?CPs?based-nerve guide conduits enhance nerve regeneration by maintaining electrical signal pathways.However,current CPs are limited by their shortcomings such as poor solubility and processing difficulties,and are difficult to be widely used.Therefore,it is urgent to develop new peripheral nerve repair conductive materials to promote nerve regeneration.Objectives:This series of research focuses on the exploration of new conductive materials used for fabricated NGC.First,carbon nanotubes?CNTs?-doped poly??-caprolactone??PCL?was used to fabricate hollow NGC with adjustable orientation through electrospinning technique.After fully characterized the physical and chemical properties of PCL and PCL/CNTs,and their effects on the behavior of nerve cells.We verified their neural regeneration-promoting effects in vivo.Based on this hollow conduit,in the second experimental system we further developed soluble CP poly?3-thiopheneacetic acid??PTAA?and tannic acid?Ta?-doped poly?lactic acid-glycolic acid??PLGA/PTAA/Ta,PPT?electrospun fiber film.Then aligned PPT?A-PPT?electrospun fiber NGC was fabricated based on this A-PPT electrospun fiber film.This A-PPT NGC filled with Schwann cells?SCs?-loaded mPEG-PLAla-CTA temperature-sensitive electroactive hydrogel.After fully characterizing the A-PPT scaffold and mPEG-PLAla-CTA hydrogel,effects and mechanism for promoting nerve regeneration of this tissue-engineered nerve graft?TENG?in conjunction with electrical stimulation?ES?were explored.Materials and Methods:First,the PCL and PCL/CNTs electrospun fiber membranes with adjustable orientation were fabricated by adjusting the rotation speed of the rollers,and fully characterize the electrospun fiber membranes with scanning electron microscope?SEM?,water contact angle,mechanical properties,thermal behavior,and small-angle X-ray scattering?SAXS?,electrical conductivity,and in vitro tests on the neurobehavior of pheochromocytoma?PC12?cells.PCL/CNTs-1000 NGC with optimal orientation was selected for the further in vivo tests.The results of motor function,histological evaluation,electrophysiology,and gastrocnemius muscle atrophy were evaluated at 3 months after operation in a rat sciatic nerve 10 mm defect model.In the TENG experiment,poly?3-thiophene methyl acetate??PTMA?was synthesized by chemical oxidation of 3-thiopheneacetic acid monomer by anhydrous ferric chloride,and then PTAA was obtained by alkaline hydrolysis.A-PPT was prepared by a method similar to the previous experiment.The ring-opening polymerization of L-alanine N-N-carboxyanhydride was initiated by the methoxy poly?ethylene glycol?to obtain methoxy poly?ethylene glycol?-polyalanine?mPEG-PLAla?diblock copolymer,mPEG-PLAla-CTA hydrogel obtained by the condensation reaction of amino and carboxyl group between mPEG-PLAla and carboxyl-capped tetraaniline?CTA?,mPEG-PLAla-CTA hydrogel were fully characterized with proton nuclear magnetic resonance,Fourier Transform Infrared Spectroscopy,phase diagram,rheology,cyclic voltammetry?CV?,and degradation both in vivo and in vitro.The cytotoxicity and adhesion of SCs and proliferation experiments were also performed.The TENG and ES in vivo experiments evaluated motor regeneration,histological evaluation,neuroelectrophysiology,and gastrocnemius muscle atrophy at 3 months after surgery.The immunofluorescence semi-quantitative results of neurofilament-200?NF200?,myelin basic protein?MBP?,glial fibrillary acidic portein?GFAP?,?3-Tubulin?TUJ-1?protein expression were used to analyze its mechanism for promoting nerve regeneration.Results:An electrically conductive PCL/CNTs-1000 NGC with adjustable porosity and good mechanical properties was successfully prepared in this study.In vitro experiments confirmed that it guide the directional adhesion and proliferation of PC12 cells.The results of motor function evaluation 3 months after operation were as follows:the sciatic functional index?SFI?scores of the PCL/CNTs-1000,PCL/CNTs-0+ES,and PCL/CNTs-0 groups were-56.89±10.12,-54.69±5.42,and-69.48±5.49,significantly lower than the scores of autograft group?-29.7±8.64?and the PCL/CNTs-1000+ES group?-31.00±4.98?;the thickness of axonal myelin sheath of the autograft and PCL CNTs-1000+ES groups were 0.87±0.32 and 0.68±0.24?m,respectively.The latency of compound muscle action potential?CMAP?in the PCL/CNTs-1000+ES group?1.33±0.06 ms?was compared with the autograft group?1.27±0.08 ms?,and shorter than that of the PCL/CNTs-0 group?2.01±0.10 ms?.The CMAP amplitudes of healthy male rats,autograft,and PCL/CNTs-1000+ES groups were 12.87±0.55 mV,6.82±0.92 mV,and 6.74±0.28 mV,respectively.The CMAP amplitudes of PCL/CNTs-1000+ES group was equivalent to that of the autograft group.The CMAP peak in the PCL/CNTs-1000+ES group?6.42±0.47 mV?was much higher than the PCL/CNTs-1000 group?4.27±0.28 mV?and PCL/CNTs-0group?3.48±0.77 mV?;gastrocnemius muscle atrophy evaluation results showed no statistical difference between the CL/CNTs-1000+ES group and the autograft group,and the proportion of cross-sectional muscle fiber area was not different from the autotransplantation group;NF200,MBP The results of semi-quantitative analysis of fluorescence intensity of GFAP and TuJ-1 staining proteins showed that the expression level of the protein in the PCL/CNTs-1000+ES scaffold was significantly higher than that of other groups,and there was no difference from the autograft group.In the second experiment,the prepared A-PPT electrospun fiber membrane had good orientation and good hydrophilicity.The electrical conductivity of the A-PPT electrospun membrane in the orientation direction was?0.21±0.06?×10^-3 S/m,the electrical conductivity perpendicular to the orientation direction was?0.08±0.02?×10^-4 S/m;the conductivity of random direction PLGA/PTAA/Ta?R-PPT?electrospun membrane was?0.32±0.09?×10^-4 S/m;the prepared lumen filling mPEG-PLAla-CTA hydrogel had good gelling performance,rheological tests showed that its storage modulus was higher than 100 Pa,and the CV curve showed that it had stable electrical activity;In vitro degradation experiments mPEG-PLAla-CTA degraded to 51.7±2.1%,35.0±2.6%,19.3±3.8%of the original weight after 30days in PBS,elastase and chymotrypsin,respectively.No obvious inflammatory response had been appeared in in vivo degradation experiments until 4 weeks;in vitro cell experiments showed that the electroactive hydrogel and A-PPT electrospun membrane simultaneously with ES showed no significant cytotoxicity to SCs,and could promote SCs adhesion and proliferation.The SFI scores of the Autograft group,A-PPT-Gel-SCs-ES group,and A-PPT-Gel-SCs group in the evaluation of motor function in animal experiments were-36.30±3.67,-38.12±4.18,-42.86±1.62,there were no statistically significant difference in the results of these three group,indicating that the recovery of motor function in the A-PPT-Gel-SCs-ES group,the A-PPT-Gel-SCs group and the Autograft group was equivalent at 3 months after operation;the results of regenerative nerve transmission electron microscopy analysis showed that the number of regenerative axons in the 2,500?m² region size in the Autograft group and the A-PPT-Gel-SCs-ES group,the average values were 101.2 and96.0,respectively,and there was no statistical difference between the two groups;the fibers diameter statistical results of Autograft group and A-PPT-Gel-SCs-ES group with myelinated axon were 4.8±0.5?m,4.2±0.4?m,respectively.There was no statistical difference between the two groups;results of myelin thickness in the Autograft group is 1.1±0.2?m,and the A-PPT-Gel-SCs-ES group was 0.8±0.1?m,there was no statistical difference.The electrophysiological results showed that the amplitudes of CAMP in the Autograft group and A-PPT-Gel-SCs-ES group were 6.89±0.15 mV and 6.79±0.10 mV,respectively.There was no statistical difference between the two groups;the results of CAMP latency of Autograft group,A-PPT-Gel-SCs-ES group were 1.26±0.09 ms and 1.27±0.05 ms,respectively,there is no statistical difference between the two groups;the results of gastrocnemius muscle evaluation were as follows,the weight ratio results of right gastrocnemius muscle and healthy gastrocnemius muscle in the Autograft group and A-PPT-Gel-SCs-ES group were 60.5±2.9%and 64.2±2.6%,respectively.There was no statistical difference between the two results;the ratio of cross-section collagen to muscle fibers in Masson-stained images was found in the Autograft group and the A-PPT-Gel-SCs-ES group with the smallest ratios,and the average values were 9.3%and 11.3%,respectively.There was no statistical difference between the two groups;NF200,MBP,GFAP,and TuJ-1 stained proteins were semi-quantitatively analyzed for fluorescence intensity.The results showed that the expression level of protein in the PCL/CNTs-1000+ES scaffold and the autograft group were significantly higher than those of the other groups,and there was no difference between the two PCL/CNTs-1000+ES scaffold and the autograft groups.Conclusions:The PCL/CNTs-1000 NGC and the TENG containing A-PPT NGC filled with SCs-loaded mPEG-PLAla-CTA temperature-sensitive conductive hydrogel were doveloped in our series of studies.for and exogenous ES.The results of these conductive NGC/TENG with ES in SFI scores,CAMP amplitudes and latencies,axon and myelin thickness,gastrocnemius muscle atrophy and other results were equivalent to autograft group in rat sciatic nerve defect experiments.,both could promote the expression of nerve-specific proteins NF200,MBP,GFAP and TuJ-1,indicating that this series of conductive NGC/TENG combined with ES could effectively target the pathological characteristics of peripheral defects,oriented NGCs achieved axon regeneration and myelination through physical guidance.The degradation rate of mPEG-PLAla-CTA thermosensitive conductive hydrogel was matched with nerve regeneration,which could provide physical support in the early stage after peripheral nerve injury,and the space left after degradation provided space for regenerating nerves.Cooperating with exogenous ES further improved the nerve repair effect of this series of NGC/TENG,and realized the precise repair of nerve function in peripheral nerve motor function,histology and electrophysiology.The development of TENG containing A-PPT NGC filled with SCs-loaded mPEG-PLAla-CTA temperature-sensitive conductive hydrogel is expected to increase the limit repair length and eventually replace autograft nerve transplantation.The successful implementation of this series of studies will provide a class of multifunctional TENGs that can repair peripheral nerve defects of critical length,which will have positive economic and social benefits.