On The Emulsion Electrospun Tissue Engineering Scaffolds For Nerve Regeneration

Emulsion electrospun core-shell composite nanofiber mats can mimic the architecture of natural extracellular matrix,thus provides the adhesion site for cells and support cell growth.Additionally,such kind of composite nanofiber mats can also be used to encapsulate(protect)watersoluble drugs,protein,and bioactive growth factors.Therefore,drug loaded composite nanofiber mats combine the characteristics of drug release system and tissue engineering scaffolds,which helps in tumor cell inhibition,cell growth/differentiation,and tissue reconstruction.Recently,researchers in emulsion electrospinning field have been simply focused on the usage of new polymers,new surfactant/emulsifier or new drugs.Some achievements have gained from those studies,but how do the emulsion factors influence the physicochemical and encapsulation/release properties of the emulsion electrospun drug-loaded nanofibers remains a question.In order to utilize emulsion electrospinning technique more effective and prepare optimal coreshell nanofibrous scaffolds for nerve regeneration,the optimization research was conducted via emulsifier/surfactant,polymer matrix,water phase,and water/oil ratio selection.Such that,coreshell nanofibers loaded with drugs,protein or nerve growth factor were well produced,and the drug release properties and biocompatibility of those composite nanofibers were studied.The potential application of NGF loaded core-shell nanofiber mats in peripheral nerve regeneration was also evaluated.Our major works are as follows:(1)In order to produce nanofibers with optimal surface morphology and physicochemical properties,we screened the most suitable emulsifier/surfactant type and concentration by single factor experiment method,and these emulsion factors are of paramount importance during emulsion electrospinning.Bovine serum albumin(BSA)was used as the model drug,while poly(?-caprolactone)(PCL)was chosen as the polymer matrix.The influence of surfactants towards the solution properties,fiber morphology,along with the physicochemical properties of the PCL-BSA nanofibers were investigated by using non-ionic surfactant sorbitan monooleate(Span80),anionic sodium dodecyl sulfate(SDS),cationic benzyltriethylammonium chloride(TEBAC)and poly(ethylene oxide)-poly(propylene oxide)–poly(ethylene oxide)triblock copolymer Pluronic F108.Results showed that with the addition of small amount of ionic surfactants/emulsifier(TEBAC or SDS),the conductivity of electrospinning solution was enhanced,and the existence of non-ionic emulsifiers(Span 80 or Pluronic F108)won't change the solution conductivity.Field emission scanning electron microscopy(FESEM)images revealed that beaded fibers were produced at low PCL concentration(8 %).However,bead-free and uniform nanofibers were produced with the incorporation of smaller amounts of emulsifier/surfactants(0.4% ~ 1%).Among the four emulsifiers,electrospinning of an emulsion containing 0.4%(w/v)SDS produced the smallest and the most uniform nanofibers(167 ± 39 nm),which was attributed to the high conductivity of the solution,followed by emulsion with 1%(v/v)Span 80.Wettability test showed that except for the TEBAC contained nanofibrous scaffolds,the hydrophilicity of Span80,F108,and SDS contained nanofiber mats improved tremendously.As the concentration of emulsifier increased,the degradation rate of PCL-BSA nanofibers got accelerated.DSC test demonstrated that all samples had a similar thermal behavior such that they showed a single peak within a relatively narrow temperature range(60.1~65.3 °C),and a melting temperature(Tm)of 60.1 °C was observed for pure PCL fibers,therefore,the incorporation of small amounts of surfactants would not significantly change the thermal properties of the emulsion electrospun nanofiber mats.The FTIR curves revealed more or less similar peaks with identical peak intensity,which means there is no formation of new chemical bonds between emulsifier and polymer.The mechanical test showed that the 0.4%(w/v)SDS incorporated nanofiber mats revealed the best comprehensive mechanical properties due to the fiber morphology,followed by the 1%(v/v)Span80 contained nanofiber mats.Our results demonstrated that with the addition of surfactants/emulsifiers,the morphological and physicochemical properties of nanofibers can be modulated.Emulsion electrospinning technique assists to produce drug loaded composite nanofibers with optimum morphology and physicochemical properties from less concentrated polymer solutions,and the scaffolds have the potential for drug delivery and tissue engineering applications.(2)To understand the influence of polymer matrix(oil phase)on physicochemical properties,drug encapsulation/release behaviors and in vitro biocompatibility of the emulsion electrospun drugloaded nanofibers,two kinds of polymers [poly(?-caprolactone)(PCL)and poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid)(PHBV)],which are different in molecular weight and physicochemical properties were selected as matrix materials.Two different water soluble drugs: metformin hydrochloride(MH)and metoprolol tartrate(MPT)were chosen as the model drugs.Nanofibrous scaffolds were produced by emulsion electrospinning of either MH or MPT with PCL or PHBV.The influence of oil phase towards the drug release behavior of the scaffolds,together with their surface morphology,physicochemical properties,and biocompatibility were evaluated.From the FESEM images we can find out that:(1)Pure PCL nanofibers appeared thinner and more uniform than the pure PHBV nanofibers,and the same applies to both drug incorporated PCL and PHBV nanofibers.This is because the molecular weight of PHBV is much higher than PCL,and the concentration of PHBV used in the experiment was nearly two times higher than that of PCL;(2)with the addition of MH or MPT,the conductivity of polymer solutions was increased,therefore,smaller and more uniform drug-loaded nanofibers were prepared compared with pure polymeric solutions;(3)as MH is a hydrochloride salt with high charge density,thinner and finer MH loaded nanofibers were produced compared with the MPT embedded nanofibers.The FTIR spectrum and DSC thermograms demonstrated that no chemical bond formed between drugs and polymers during the preparation of nanofibers,and the incorporation of small amounts of drugs and emulsifier/surfactants did not significantly change the thermal properties of the drug loaded emulsion electrospun mats.As the average diameter of the PHBV-drug nanofibers were much larger than the drug loaded PCL nanofibers,a lower density of Span 80 molecules were distributed on the surface of drug loaded PHBV nanofibers,thus leading to a lower wettability of the emulsion electrospun drug loaded PHBV nanofibers compared to emulsion electrospun drug loaded PCL nanofibers.In vitro release study revealed that the emulsion electrospun PHBV-drug nanofibers showed higher burst release and faster drug release rate than emulsion electrospun drug embedded PCL nanofibers,which could be attributed to the different inherent properties of PHBV and PCL.(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)assays showed that all the emulsion electrospun drug-loaded nanofibers have no cytotoxicity against human Mesenchymal Stem Cells(hMSCs),and among all the nanofibrous scaffolds,PCLMPT(E)nanofibers favour the survival of hMSCs.Our results suggest that the physicochemical properties and drug release rate of the nanofibers could be modulated by adjusting the oil phase(polymer matrix material),and PCL could be a better matrix for drug delivery system than PHBV.(3)A systematic research was carried on the influence of water phase properties and water/oil(W/O)ratio on the physicochemical properties and drug encapsulation/release behaviors of small molecular weight drug and macromolecule protein incorporated emulsion electrospun composite nanofibers.In this study,metoprolol tartrate(MPT)and bovine serum albumin(BSA)were chosen as small molecular model drug and macromolecule model protein,respectively.PCL was chosen as the polymer matrix.The influence of solute molecular weight,solute concentration and W/O ratio on drug/protein early release behaviors were studied.The emulsion stability test showed that:(1)the stability of emulsion added with small molecule drug MPT was worse than that of the BSA-loaded emulsion,as the ionization ability of MPT aqueous solution was stronger,which was unfavorable to the stability of emulsion;(2)increasing the solute concentration and the volume ratio of water phase/oil phase reduced the stability of emulsion and accelerated the speed of demulsification.This is because emulsion is a thermodynamically unstable system,and demulsification is an inevitability,but the demulsification time varies from the concentration and volume of dispersion phase.FESEM test results revealed that:(1)the molecular weight of the water phase solute nearly has no influence on fiber diameter;(2)increasing the concentration of water phase solute,the fiber diameter slightly increased,but the difference of sample diameter was not obvious;(3)when the W/O ratio is increased,the fiber diameter and diameter distribution significantly also increased,non-uniform,branched,and glued nanofibers were produced.Because increasing the W/O ratio,namely increase the volume of distilled water(H2O)in the emulsions.Since H2 O is not an electrospinnable solution,a higher volume of H2 O can lead to the decrease of polymeric solution spinnability,therefore bigger fibers were produced.In addition,as the evaporation speed of H2 O is much slower than that of the organic solvent,nanofibers were still wet when they reached the collector,so fiber defects such as branches and fiber bundles were observed,leading to the formation of non-uniform nanofibers.In vitro release study showed that:(1)drug encapsulation efficiency would not be affected by the molecular weight of water phase solute,while the initial release percentage will decrease when the drug size is increased.As bigger drug size can alleviate burst release and contribute to the sustained release of the drug;(2)either increase the solute concentration or water/oil ratio,the initial burst release of drugs increased,while the encapsulation efficiency decreased.The reasons for this phenomenon are as follows: firstly,the charge density of electrospinning solution was obviously increased when the concentration of water phase solute(MPT or BSA)was increased,thus aggravate jet whipping instability during the electrospinning process.A lot of drugs were excluded to the surface of the nanofibers instead of the inner site of fiber,due to the increased coulomb repulsion,leading to the burst release and lower encapsulation efficiency of the drug.Secondly,as discussed above,since the electrospinnability of H2 O is quite low,increase the volume of H2 O can lead to the decrease of polymeric solution spinnability.Accordingly,there will be a higher burst release of drug-loaded nanofibers.In order to get the ideal drug release and encapsulation efficiency results,we need to decrease the concentration of water phase solute and the W/O ratio as much as possible.Although this may result in a decrease of the drug-loading rate,the greatest advantage of electrospinning technique is the maximizing utilization of drug efficiency at low drug loading conditions.(4)To evaluate the potential application of emulsion electrospun composite nanofibers in nerve tissue engineering.Bovine serum albumin(BSA)and nerve growth factor(NGF)were both incorporated inside emulsion electrospun composite nanofibers based on the experimental results of the former three parts.The bioactivity retention extent of the encapsulated macromolecules during electrospinning process and the release behaviors of BSA/NGF from random/aligned nanofibers were studied.Rat pheochromocytoma cells(PC12 cells)was utilized to evaluate the bioactivity of NGF released from the NGF embedded nanofibers.Our results demonstrated that all the scaffolds have good biocompatibility and had no side effects on the survival of PC12 cells,while there were more live cells on(R/A)-PCL-NGF and(R/A)-PCL-NGF&BSA scaffolds compared to other scaffolds.The sustained release of a small amount of NGF from(R/A)-PCL-NGF&BSA scaffolds was enough to induce the differentiation of PC12 cells.Cells seeded on aligned PCL-NGF nanofibers showed more neurites and grew along the long axis direction of the fiber,indicating that the aligned nanofibers are favorable for the adhesion,differentiation,and migration of PC12 cells.NGF released from the PCL-NGF nanofibers were more effective on PC12 cells differentiation than that of NGF directly added in the culture medium.The topographical cue of aligned nanofibrous scaffold had a positive effect on PC12 cells differentiation.Our researchers showed that the NGF&BSA incorporated core-shell nanofibrous scaffolds could create a cell-favourable microenvironment and provide cell growth guidance,which not only can mimic the natural ECM but also can serve as a model drug delivery system for the sustained release of NGF.This optimized nerve tissue-engineering scaffold could contribute to the design of artificial nerve conduits for nerve injury repair.