The Fabrication And Application Of Microencapsulated Chitosan, Collagen/Chitosan–based Microsphere–Scaffold Delivery System For Promoting Peripheral Nerve Regeneration

Peripheral nerve defect results in partial or total loss of motor, sensory andautonomic functions in the involved segments of the body and typically lead tolife-long disability of many patients. Thus, it greatly compromises the patients’quality of life and has a significant socioeconomic impact. End-to-endcoaptation and autologous nerve grafts are widely used technique for smallnerve gaps. Nevertheless, they have several inherent disadvantages andlimitations. To overcome the problems, different types of artificial nerveconduits have been explored as alternatives to autografts to repair peripheralnerve defect. Although the FDA (Food and Drug Administration) have alreadyapproved several scaffolds based on natural and synthetic biomaterials to repairperipheral nerve defect, including NeurotubeTMPGA and NeuraGenTMCollagen, the clinical and experimental outcomes of these empty nerve scaffolds forperipheral nerve repair remain unsatisfactory. The limited success of thesehollow scaffolds may be attributed to the lack of efficient inner microstructureand neurotrophic supports for guiding the growth of regenerated nerve.The inner microstructural properties of scaffold have been reported tosignificantly affect its performance in guiding nerve regeneration. Suchscaffolds have been demonstrated to facilitate better peripheral nerveregeneration than hollow or random inner structural scaffolds. Previously, ourlaboratory has fabricated a novel nerve-guiding collagen-chitosan (CCH)scaffold with inner dimensions resembling the basal lamina micro-channels ofnormal nerves. The micro-channels in CCH scaffold were capable of guiding thelinear growth of regenerating axons. However, the CCH scaffold lack trophiccue, which is another important factor in promoting nerve regeneration.Therefore, incorporation neurotrophic factors into CCH scaffold may be apromising strategy for improving nerve regeneration. Neurotrophic factor can bephysical absorbed into a polymeric scaffold to be used for nerve regeneration.The release of these factors is then controlled by diffusion and/or scaffolderosion or degradation mechanisms. However, a general problem in using thismethod includes undesired initial burst release and limited bioactivity. Therefore,it is necessary to develop a protein drug delivery system for improving thebiological utilization of neurotrophic factor and protecting its bioactivity fromdegradation by direct exposure to harsh environments, e.g., light, oxygen,chemicals, etc.The chitosan microspheres loaded with nerve growth factor (NGF–CMSs)were prepared by the emulsion-ionic cross-linking method with sodiumtripolyphosphate (STPP) as an ionic cross-linking agent. In vitro release tests and bioactivity assay suggested that NGF–CMSs were capable of releasingbioactive NGF over7days, thus having potential application in nerve injuryrepair. Microencapsulated chitosan, collagen/chitosan–based microsphere–scaffold delivery system (NGF–CMSs/CCH) was fabricated by incorporatingNGF–CMSs into CCH scaffold using a post-seeding technique.NGF–CMSs/CCH had longitudinally oriented microchannels withinterconnected porous structure. Moreover, NGF–CMSs were distributedthroughout the microchannels which did not influence the structures ofNGF–CMSs/CCH. NGF was delivered from NGF–CMSs/CCH in a temporallycontrolled manner, depending on the degradation of both NGF–CMSsincorporated and CCH matrix. NGF–CMSs/CCH was used to bridge a15mmexcision of the sciatic nerve. The in vivo animal study demonstrated thatNGF–CMSs/CCH group promoted axonal regeneration and functional recoverywith a satisfactory efficacy close to that by autograft and better than that byimplantation with other scaffold groups. The whole study consisted of two parts:PartⅠ: Ionically cross-linked chitosan microspheres for controlled releaseof bioactive nerve growth factor[Objectives] To investigate the feasibility of the controlled release of bioactivenerve growth factor (NGF) with ionically cross-linked chitosan microspheres(NGF–CMSs).[Methods] The microspheres were prepared by the emulsion-ionic cross-linkingmethod with sodium tripolyphosphate (STPP) as an ionic cross-linking agent.The size and distribution of the microspheres, SEM images, Fourier transforminfra red spectroscopy (FTIR), Encapsulation efficiency, in vitro release tests and bioactivity assay were subsequently evaluated.[Results] The microspheres had relatively rough surfaces with mean sizesbetween20and31μm. FTIR results provided evidence of ionic interactionbetween amino groups and phosphoric groups of chitosan and STPP. The NGFencapsulation efficiency ranged from63%to88%depending on theconcentration of STPP. The in vitro release profiles of NGF from NGF–CMSswere influenced by the concentration of STPP. NGF–CMSs which werecross-linked with higher concentration of STPP showed slower but sustainedrelease of NGF. In addition, the released NGF from NGF–CMSs was capable ofmaintaining the viability of PC12cells, as well as promoting theirdifferentiation.[Conclusions] NGF–CMSs are capable of releasing bioactive NGF over7days,thus having potential application in nerve injury repair.Part Ⅱ: Repair of Peripheral Nerve Defect Using MicroencapsulatedChitosan, Collagen/Chitosan–Based Microsphere–Scaffold Delivery System[Objectives] Nerve repair ability of microencapsulated chitosan,collagen/chitosan–based microsphere–scaffold delivery system was investigatedin present research.[Methods] A novel microsphere–scaffold delivery system was fabricated byincorporating chitosan microsphere encapsulated nerve growth factor(NGF–CMSs) into collagen–chitosan (CCH) scaffold using a post-seedingtechnique. Physical absorbed NGF into CCH scaffold (NGF/CCH scaffold),unloaded chitosan microsphere incorporated into CCH scaffold (CMSs/CCHscaffold) and CCH scaffold were used as control group. SEM images, in vitro release tests and bioactivity assay were evaluated. Afterward, the CCH,NGF/CCH, or NGF–CMSs/CCH group was used to bridge the nerve defect. Atthe4,8, and12weeks end points, electrophysiological tests, fluorogoldretrograde tracing, walking track analysis and histological investigation weresubsequently evaluated.[Results] Compared with rapid release from CMSs/CCH or NGF/CCH group,NGF was delivered from NGF–CMSs/CCH microspheres-scaffold composite ina temporally controlled manner, depending on the degradation of bothNGF–CMSs incorporated and CCH matrix. PC12cells were co-cultured withCCH, CMSs/CCH, NGF/CCH, and NGF–CMSs/CCH group, respectively, andin vitro bioactivity results indicated that, with the appearance of NGF–CMSsinto microspheres-scaffold composite, the PC12cells exhibited better cellviability. The in vivo animal study demonstrated that NGF–CMSs/CCH grouppromoted axonal regeneration and functional recovery with a satisfactoryefficacy close to that by autograft and better than that by implantation with CCHor NGF/CCH group.[Conclusions] The promising NGF–CMSs/CCH microspheres–scaffoldcomposite may be used as ideal delivery system for peripheral nerveregeneration.