Fabrication Of Bioactive Neural Tissue Engineering Scaffolds Incorporatting Dual-neurotrophic Factors By Coaxial Electro Spinning

Engineering scaffolds,cell sources and growth factors are the three elements of tissue engineering(TE),developing bioactive substitutes for the repair and regeneration of damaged tissues based on these three elements which can closely mimic the componential and structural aspects of the native extracellular matrix is a core task and great challenge in tissue engineering field.Mainly caused by the trauma from accidents,peripheral nerve injury is a common clinical disease.Among the performed peripheral nerve surgeries,direct suture and substitutional nerves from other parts of the body are also widely used,both of which have some defects.Neural scaffolds combined with neurotrophic factors and cell sources accelerate the research of long gap nerve defects repair.However,the common method of neurotrophic factors combining by "physical adsorption" is not ideal,therefore,to prepare a neural scaffolds incorporatting neurotrophic factors which can control release behavior and protect the biological activity of the neurotrophic factors is a essential work to neural tissue engineering syudy.In this study,we incorporated monosialoganglioside GM1(GM1)and nerve growth factor(NGF)into nanofibrous scaffolds which can protect their biological activity by coaxial electrospinning,also,we get the aligned nanofibrous scaffolds by a high rotating drum to mimic the structure of nonnal nerve basement membrane.These electrospun scaffolds have a high surface-to-volume ration,a high porosity,aligned nanofibers and bioactive neurotrophic factors.Scanning electronic microscope(SEM)and transmission electron microscopy(TEM)revealed that the scaffold was composed of aligned nanofibers and apparent continuous "core-shell" structure respectively,the TEM images also exhibited there was a smooth core-shell interfaces between the core phase containing GM1/NGF and the shell phase of Silk fibroin(SF)and P(LLA-CL).The contact angle tests showed that silk fibroin can inchance the hydrophilic propertie of the scaffold,in addition,the mechanical test demonstrated that the nanofibrous scaffold had excellent mechanical properties,and the tensile strength was much higher at parallel than at perpendicular directions.In vitro release of neurotrophic factors was analyzed using Duoset ELISA kit,and the results was presented in terms of cumulative release.The release profiles showed that after a initial burst release,GM1 and NGF both can be released in a relatively steady manner,no matter from single component neurotrophic factor scaffold or two components neurotrophic factors scaffold.Then,the Pheochromocytoma cells(PC 12)morphological change of differentiation and Schwann cells(SCs)proliferation test demonstrated that NGF and GM1 released from nanofibers at least remain part of their biological activity.SCs and PC 12 cells were utilized as cell sources to examine the biocompatibility of the nanofibrous scaffolds.MTT assay showed nanofibrous scaffolds incorporating GM1 and NGF supported cell proliferation better than the scaffolds only incorporating NGF,also,in terms of cell proliferation,the aligned structure had a positive effect.Both the SEM and confocal laser scanning microscope(CLSM)images confirmed that the cells spread out well on the aligned nanofibrous scaffolds,SCs elongated along the fiber axes and emulated the structure of bands of Bungner on the aligned nanofibrous scaffolds,more axons and longed axons of PC 12 were appeared on the GM1-NGF nanofibrous scaffolds compared with the NGF nanofibrous scaffolds,and the aligned nanofibers provided a guide for regenerating axons.