| Objective:(1)To fabricate multi-channel oriented composite neural conduits with various chitosan and collagen(CH-CO)mass ratios,to characterize their physical and mechanical stability,to assess their biocompatibility through diverse methods,and to ultimately select the conduits with the optimal mass ratio for in vivo experiments based on their superior performance.(2)To synthesize PLGA microspheres loaded with Glial Cell Line-Derived Neurotrophic Factor(GDNF),and to further develop carboxymethylated chitosan(CSMA)/PLGA composite microspheres(CP-MS)that encapsulate these microspheres.This process involves characterizing the general properties of the microspheres,verifying their biocompatibility,and assessing their drug loading and release capabilities.(3)To integrate the specifically selected CH-CO mass ratio conduits with uniformly sized and well-dispersed CP-MS for the repair of sciatic nerve defects in rats.This step aims to further explore and evaluate the regenerative effects of the composite neural conduits combined with CP-MS microspheres on rat sciatic nerve regeneration.Method:(1)Fabrication of Neural Conduits: Utilizing electrospinning,the outer sheath of PLGA was fabricated,followed by the creation of the inner multi-channel CH-CO conduits through directional freezing.CH-CO was prepared in mass ratios of 4: 1,1: 1,and 1: 4.These conduits were characterized by observing microstructures,measuring average pore sizes,porosity,degradation rates,compressive strength of the conduits,and tensile strength of the PLGA sheaths.Biocompatibility was assessed through CCK-8 cell proliferation assays,live/dead cell staining after 3D cell culture,flow cytometric apoptosis assays,and co-culturing with PC12 cells.The impact of different CH-CO mass ratios on the proliferation and growth of PC12 or RSC96 cells was evaluated in vitro to select the neural conduit with the best overall performance for in vivo studies.(2)Preparation of Microspheres: GDNF-loaded PLGA microspheres were produced using an emulsion solvent evaporation method,followed by the creation of CSMA/PLGA composite microspheres(CP-MS)through microfluidic technology,photo-crosslinking,and Na OH solidification.The surface and structure of PLGA microspheres and CP-MS were examined using Scanning Electron Microscopy(SEM)and laser confocal microscopy.The uniformity and dispersion stability of CP-MS under different microfluidic aqueous and oil phases were analyzed under an optical microscope.The degree of CSMA methacrylation,encapsulation efficiency,and drug loading were determined by Nuclear Magnetic Resonance(NMR)and ELISA,respectively.The release of GDNF from CP-MS was quantified at predetermined intervals by ELISA.The biological activity of CP-MS was evaluated by inducing differentiation in undifferentiated PC12 cells and assessing biocompatibility through CCK-8 proliferation assays and live/dead cell staining.(3)In Vivo Implantation: The selected CH-CO/PLGA neural conduits and prepared CP-MS were implanted together into a 10 mm sciatic nerve defect model in rats.The animals were divided into four groups: autologous nerve transplantation(ANT),multi-channel neural conduit combined with CP-MS(POC-GLM),multi-channel neural conduit combined with GDNF(POCG),and the multi-channel neural conduit alone(POC).The effectiveness of peripheral nerve regeneration was comprehensively evaluated using color Doppler ultrasound,rat footprint analysis,Sciatic Functional Index(SFI),nerve electrophysiology,retrograde tracing with adeno-associated virus r AAV-Retro(e GFP),histological examination of regenerated nerves,wet muscle weight measurement,and histological analysis of the gastrocnemius muscle.Results:(1)The CH-CO(1: 1)/PLGA conduits displayed a directional pore structure with an average pore diameter of 47.6±5.4 μm,conducive to PC12 cell growth,and exhibited no significant difference in porosity across different mass ratios.Degradation tests with 0.1% collagenase over 21 days showed stable degradation rates for CH-CO(4: 1)and CH-CO(1: 1)conduits at 19.4±3.2% and29.6±4.7%,respectively.Biocompatibility tests indicated enhanced cell proliferation and growth,particularly for CH-CO(1: 1)and CH-CO(1: 4)ratios.Mechanical testing revealed that the CH-CO(1: 1)/PLGA conduits maintained substantial integrity post-compression,with minimal reduction in maximum stress(16.6%)and diameter(19.5%).The PLGA sheath demonstrated significant support for RSC96 cell adhesion and proliferation,evidencing good biocompatibility.(2)GDNF-loaded PLGA microspheres had an average diameter of 7.2±3.8 μm.Adjusting the microfluidic flow rates achieved uniform CP-MS sizes ranging from 145.7±21.3 μm to 52.1±4.1 μm,with optimal dispersion and structure integrity.SEM and confocal microscopy confirmed the smooth,spherical surface of PLGA microspheres and the distinct core-shell architecture of CP-MS.ELISA analyses revealed an encapsulation efficiency of 81.2±1.9% and a drug loading of0.84±0.11%.The release profile of GDNF from CP-MS indicated sustained biological activity up to six weeks,promoting differentiation in PC12 cells without impacting cell proliferation,as compared to controls.(3)Two weeks post-implantation of CH-CO(1: 1)/PLGA conduits and CP-MS into rat sciatic nerve defects,Doppler ultrasound confirmed the continuity of regenerating nerves without conduit collapse.Ten-week follow-ups showed no narrowing or twisting at the nerve graft sites,indicating smooth,continuous nerve integration.Comparative analyses at 12 weeks post-surgery showed that POC-GLM group’s myelinated nerve fiber density and diameter were close to those of the ANT group,with no significant differences in myelin sheath thickness.Immunofluorescence for S100,NF200,MBP,and GAP-43 proteins showed that the expressions in the POC-GLM group were significantly higher than in the POC group and comparable to the ANT group,suggesting effective nerve regeneration and functional recovery.Conclusion:(1)CH-CO(1: 1)/PLGA conduits exhibited a directional porous structure,with pore sizes conducive to the growth of PC12 cells,high porosity,and stable degradation properties.They demonstrated exceptional mechanical properties in both compression and tensile tests,alongside superior biocompatibility.The PLGA sheath significantly supported the proliferation,adhesion,and growth of RSC96 cells,meeting the requirements for in vivo implantation experiments.(2)The CP-MS showed uniform size and dispersion with a distinct core-shell structure.They exhibited high encapsulation efficiency and drug loading capacity,successfully inducing differentiation in PC12 cells and maintaining biological activity for up to six weeks.The CP-MS demonstrated excellent biocompatibility,with no adverse effects on the proliferation and growth of PC12 cells,underscoring their potential in peripheral nerve regeneration applications.(3)The combination significantly enhanced nerve regeneration,myelination,and the restoration of nerve and muscle function in vivo.The directional multi-channel structure provided physical guidance and support,while the composite microspheres extended the release duration of GDNF,collectively facilitating the regeneration process of peripheral nerves. |
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