| Peripheral nerve injury,as a worldwide clinical problem,which is often caused by trauma or surgery,can lead to the partial or total loss of motor function and sensory perception,and even lifelong disability.Autologous nerve transplantation,which acts as an ideal immunogenically inert scaffold for axonal regeneration,is accepted as the "gold standard" for therapy of irreducible nerve defects.However,the autologous nerve transplantation is limited by the shortage of sources,the considerable morbidity of the donor-site,the dimensional mismatch between graft and host nerves and other potential complications.To overcome these limitations,various biomaterials and seed cells have been utilized to fabricate artificial nerve scaffold for bridging the nerve defects and have achieved varying degrees of success Among these approaches,artificial nerve composed of aligned nanofibers received wide attention.It has been proved that alignment profile could influence cell adhesion,proliferation and mediates stem cell differentiation.For nerve tissue applications,the alignment is necessary to guide the cells migration in a particular direction and promote the axonal elongation.It have been reported that when cultured on the aligned electrospun PCL fibers,Schwann cell(SC)cytoskeleton and nuclei were aligned and elongated along the fiber axes,emulating the band of Bungner,which is an important supporting structure for axonal elongation during nerve development and regeneration.As the most promising seed cells for peripheral nerve tissue engineering,SCs can synthesize and secrete various neurotrophic factors and extracellular matrix to guide and promote axonal growth.Moreover,the regenerated peripheral axons have to be remyelinated by SCs for the functional nerve conduction.Unfortunately,due to the damage of donor nerve for traditional SCs isolation,and SC's low capability of proliferation,it is difficult to obtain and expand enough SCs to the therapeutic needs.Previous study indicated that bone marrow derived mesenchymal stem cells(BMSCs)can be induced to express SC's functional proteins and the induced cells could myelinate the axons after be transplanted.Aligned nanofibers can significantly enhance the SCs transdifferentiation from BMSCs,which indicates that the topology of microenvironment play important roles in the SCs differentiation.Considering that SCs are living along with the electrical conductive axons,we hypothesized that the conductivity property may also facilitate the SCs differentiation and then improve the efficacy of nerve tissue engineering.To verify this hypothesis,the amine functionalized multi-walled carbon nanotubes(MWCNT)were incorporated with Polycaprolactone(PCL)and Gelatin to fabricate aligned or random conductive nanofibers by electrospinning.After be evaluated by scanning electron microscopy,electrochemistry workstation and the co-cultured cells live-dead staining assay,the nanofibers were used as scaffolds to culture the BMSCs,and the SCs induction was performed on it.Furthermore,a PCL conduit filled with PCL/Gelatin/MWCNT fiber membranes was prepared and seeded with the induced BMSCs(iBMSCs)to bridge a 10-mm defect of rat sciatic nerve.Methods:1.Electrospining was used to fabricate the nanofiber membranes,including aligned nanofibers(A),random nanofibers(R),aligned and conductivity nanofibers(AC),random and conductivity nanofibers(RC).The surface morphology of the electrospun PCL/gelatin and PCL/gelatin/MWCNT nanofibers were evaluated by scanning electron microscope.The diameters of the electrospun fibers were measured from the image using Image J software.Cyclic voltammetry(CV)measurements were performed with an electrochemical workstation.The biocomoatibility of the membranes were assessed using live-dead staining.2.BMSCs were isolated from the femurs and tibias of 2-week old Sprague Dawley(SD)rats and seeded onto the membranes of electrospun fibers.The cells were subjected to do the transdifferentiation with our previous three steps protocol.In order to identify the efficiency of the SCs transdifferentiation,the induced cells were immunostained with specific markers of SCs,including glial fibrillary acidic protein(GFAP),Nerve growth factor(NGF)and S100 by our routine procedures.3.To assay the bioeffects of the induced BMSCs on the neurite outgrowth,we used dorsal root ganglian(DRG)as another model neuron for further study.DRGs were harvested from the spinal column in SD rat pups at the age of 1 day via sterile microdissection,and seeded onto the center of the nanofiber membranes,which have been pre-seeded with BMSCs and induced for 2 weeks in differentiation medium.After 5 days of culture,the extension of DRG axons were detected by immunofluorescence staining.4.A combined artificial nerve conduit was developed by a cylindrical conduit and AC nanofibers with induced BMSCs,and then used to bridge a long defected sciatic nerve in rat.At 12 weeks after transplantation,the expression of Neurofilament(NF)and myelin basic protein(MBP)was detected by immunofluorescence staining to determine the axonal regeneration and remyelination.Functional recovery and target tissue repair after nerve injury were determined by wet weight ratio of gastrocnemius muscle,HE staining of gastrocnemius muscle cross section and nerve electrophysiological test.Results:1.Prepartion and characterization of the Electrospun Fibers1.1 Scanning electron microscope images revealed that these fibers exhibited a smooth surface and have a range of diameter distribution from 200?900nm.1.2 Compared with the non-conductive fibers(R and A),the membranes composed of RC and AC fibers showed dramatically higher conductivity.Collectively,MWCNT can dramatically increase the electrical conductive property of the nanofibers.Therefore,the scaffolds of A,R,AC and RC fibers can be used to assess the synergistic effect of electrical conductivity and aligned alignment profile on the SCs trasndifferentiation in the following experiments.1.3 Live/dead staining was also performed to assay the potential cytotoxicity of the scaffolds.The results indicate that our prepared electrospun nanofibers had excellent biocompatibility and negligible cytotoxicity.2.Effects of fibrous membrane on BMSCs differentiation into SCs and DRG neurite elongation2.1 The aligned nano fibers can promote the differentiation of BMSCs into SCs,which is consistent with previous reports.The random nanofibers can not promote this transdifferentiation,no matter they are conductive or non-conductive.The conductivity can enhance the SCs differentiation efficiency on the aligned nanofibers.2.2 The length of DRG neurite was measured by the immunofluorescence staining of?-tubulin ?(Tuj1).The results showed that both of average length and maximal length of the neurites were ranked first in AC group,second in A group,which proved that the iBMSCs induced on the AC fiber membrane significantly promoted the growth of DRGs neurite,and there was no significant difference among R,RC and control groups.3.The role of artificial nerve conduits containing AC membrane in the regeneration of injured sciatic nerves3.1 Immunofluorescence staining showed that the iBMSCs induced on the AC membrane could differentiate into mature SCs and participate in the formation of myelin sheath.The artificial nerve conduits containing AC membrane significantly promoted axonal regeneration and remyelination after sciatic nerve injury.3.2 The results of electrophysiology and HE staining of gastrocnemius cross section demonstrated that the artificial nerve conduits containing AC membrane promoted the recovery of sciatic nerve conduction function and the target muscle innervated.Conclusions:The key objective of this study is to explore the synergistic effect of conductivity and alignment profile on the differentiation of BMSCs into SCs.Current data demonstrated that BMSC could be transdifferentiated into SCs,aligned nanofibers could improve the efficacy of the transdifferentiation while the random nanofibers had no effect on it.More importantly,we found that endowing conductive property into the aligned nanofibers could significantly enhance its capability to promote the SCs differentiation,however,the addition of electrical conductivity in the random nanofibers cannot change their negative results on the SC differentiation.Furthermore,both of in vitro and in vivo results demonstrated that the aligned and conductive nanofibers could promote peripheral axonal regeneration.Collectively,present study indicated that the conductive property in the aligned nanofiber plays significant roles in the SCs differentiation,the aligned and conductive nanofibers could be used as a promising scaffold for the peripheral nerve tissue engineering. |
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