Biological Function And Mechanism Of Bone Marrow Mesenchymal Stem Cells-packed Poly(3,4-ethylenedioxythiophene)(PEDOT)Scaffolds For Peripheral Nerve Injury:The Involvement Of MiR-21-notch Signaling Pathway

Part One The preparation of PEDOT-AMT scaffold by in-situ polymerization of PEDOT on acellularized muscle tissueABSTRACTBackgrounds:Peripheral nerve injury leads to substantial motor and sensory deficits and ultimately these deficits can eliminate an individual's ability of daily living.Unfortunately,not all of these nerve injuries can be repaired using biological tissues as auto-transplantation.Possible solutions to the limited availability of autografts include the use of chemically acellularized,non-synthetic tissue allografts.The acellularized muscle constructs are of particular interest because they are composed of cytoskeleton proteins,collagen and proteoglycans that may be derived from allogenic tissues.Elimination of cellular elements from the tissue permits use of the ECM meshwork as an allograft for implantation without the need for immunosuppression.Poly(3,4-ethylenedioxythiophene)(PEDOT)belongs to a group of conjugated polymers known for its good electrical conductivity and environmental stability.It has been shown to significantly reduce the impedance of biomedical electrodes designed for long-term implantation in living tissue.It is worth exploring that if the PEDOT-modified acellular constructs could be biologically advantageous to nerve grafts.Purpose:In this report,we aim to demonstrate the chemical in situ(withinthe tissue)polymerization of the conductive polymerPEDOT on acellular muscle tissue(AMT)constructs andcharacterize the acellular muscle tissue with polymerizedPEDOT(AMT-PEDOT)constructs using microscopy,chemical analysis,and surface analysis methods.The purpose of our work was to investigate the optimum conditionsfor the production and manufacture of biologically inactivetissue with a conductive coating.Methods:Acellularized muscle tissues(AMT)were prepared from full-thickness mouse vastus lateralis muscles using chemical acellularization method based on NaN3.In situ polymerization of EDOT was perfomed with a mild oxidizer(Fe3+)in a reaction that takes placedirectly on the AMT tissue.Samples were abundantly washed and sonicated in deionized water with repeated rinses and change of sonicated solutions to help eliminate excess iron and chlorine from the constructs.Observations of optical microscopy and scanning electron microscopy were performed to acquire the microstructure of tissue coating in different conditions(sonicated AMT,AMT+PEDOT1 and AMT+PEDOT6).X-ray photoelectronspectroscopy(XPS)measurements were performed to provide accurate information on the presence of the iron on the surface or outermost layers of the construct.The total iron in construct was measured using elemental analysis method with an Inductively Coupled Plasma-Optical Emission Spectrometer.Results:The AMT constructs were successfully prepared and had a white yellowish coloration,composed of bundles of fibers.The polymerization process resulted in the deposition of a black compound,PEDOT,on the tissue and had a dark black appearance.After polymerization,the shrinking of the tissue is readily observable by the snaked eye,and the previously pliable AMT construct became stiff and brittle.The anisotropic structure of the AMT construct,albeit with a directional preference along the fibers,results in the anisotropic structures of the polymerization.The PEDOT coatings grew thicker with increasing polymerization cycles.Comparing PEDOT1 and PEDOT6 constructs indicated that,to obtain good substrate modification and surface coverage,it was necessary to perform additional cycles of polymerization.XPS measures indicated most of the iron came from the chemical oxidation procedure and was reduced during polymerization.The sodium and chlorine peaks were disappeared after sonication.Elemental analysis of control AMT constructs gave an average iron concentration of 0.43%in percentage weight and AMT+PEDOT6 had a 3.32%iron content.This 6-fold average increase with respect to the control samples seems a fairly reasonable increase after dedoping.Conclusion:In summary,in this report,we present a method for thechemical polymerization of the conductive polymer PEDOTin acellularized AMT constructs.We determined that a goodmethod of preparing the samples for their possible use asacellular constructs for implantation in vivo was to polymerize with iron chloride as the oxidant.The polymer waspartially de-doped using sonication for 30 min in ethanol,reducing iron and chlorine from the AMT constructs toacceptable metabolic levels while preserving the structuralintegrity of the AMT construct with the polymer.We expect these results provided the basis for applications of AMT-PEDOT constructs in surgical reconstructions in peripheral nerve repairs.Part Two Biological Function and Mechanism of Bone Marrow Mesenchymal Stem Cells-packed Poly(3,4-ethylenedioxythiophene)(PEDOT)Scaffolds for Peripheral Nerve Injury:The Involvement of miR-21-Notch Signaling PathwayBackgrounds:Ununited peripheral nerves represent attractive site for connectivity with neuroprostheses because their predictable internal topography allows precise sorting of motor and sensory signals.Also transplantation of bone marrow mesenchymal stem cells(BMSCs)is increasingly recognized as an effective method of restore the peripheral nervous system injury due to its neuron-directed differentiation potential.MicroRNAs(miRs)attract researchers' attention for its regulatory role in differential expression of BMSCs genome,which is essential for multiple differentiation of BMSCs.Notch signaling pathway is well documented in mediating neuronal differentiation of BMSCs and is closely related to the Schwann cell function and repair of peripheral nerve.However,no evidence focusing on the functional interaction of miR and Notch pathway in regulating BMSCs-derived neural differentiation has been reported.Purpose:This study was to evaluate the in vivo performance of BMSCs-packed Poly(3,4-ethylenedioxythiophene)(PEDOT)scaffolds across a critical nerve conduction gap and examine the potential mechanism by which BMSCs-packed PEDOT scaffolds mediate peripheral nerve regeneration in rat model of sciatic nerve transection.Methods:In this study,rat models of 8 mm sciatic nerve deletion were randomly divided into three groups and respectively received transplantation of autologous nerve or PEDOT scaffolds or BMSCs-packed PEDOT scaffolds.After transplantation,neurological function was assessed by electro-myography.BMSCs-directed neuron differentiation was induced and confirmed by the upregulation of 6 nerve cell markers.Expression level of miRs,Notch signals and 6 nerve cell markers in nerve grafts or cells were determined by quantitative real-time PCR or western blot analysis.Cellswere then pre-treated with Jagged-1/Fc(an agonist for Notch 1 pathway)or GSI(an antagonist for Notch 1 pathway),then in vitro induction of BMSCs-derived neuron-like differentiation was carried out and changes of 6 nerve cell markers were re-measured to determine the involvement of Notch pathway in this process.The procedure was repeated by transfecting cells with miR-21 mimic or miR-21 inhibitor to determine the impact of manipulation of miR-21 on this process.Results:The results showed that BMSCs-packed PEDOT scaffolds transplantation significantly improved neurological function compared with PEDOT scaffolds alone.Regenerative nerve of BMSCs-packed PEDOT scaffolds showed higher expression level of miR-21 and Notch signals(Hes-1 and Notch intracellular domain(NICD))than PEDOT alone.We found that the expression levels of miR-21,Hes-1 and NICD are increased with time-course of neuron-directed differentiation stimulating of BMSCs.Meanwhile,both miR-21 overexpression and Notch pathway activation promote the expression of 6 nerve cell markers in BMSCs-directed neuron,whereas the inactivation of Notch pathway abrogates miR-21-inudced upregulation of 6 nerve cell markers.Moreover,knock-down of miR-21 suppresses the pro-neural restoration action of BMSCs-packed PEDOT scaffolds.Conclusion:In summary,our data suggested that BMSCs-packed PEDOT scaffolds effectively repairs sciatic nerve injury.It was also illustrated that functional upregulation or activation of miR-21 or Notch signaling pathway during neuronal induction of BMSCs and come into being a mechanism for the effect of scaffolds on repair process.