Electrospun Highly Aligned Ultrafine Fibers Of Gr/PLLA Composite For Nerve Conduit Fabrication

Electrospun ultrafine fibers in nano-/micro-scaled fineness that mimic the composition and structure of natural extracellular matrix(ECM)hold a great promise in the field of tissue engineering and regeneration medicine.However,the fibers produced from conventional electrospinning(CES)technique usually have inferior orientation degree,leading to poor guidance in oriented growth and differentiation of cells while engineering anisotropic tissues(e.g.,nerve,tendon).Nerve conduits(NCs)are usually constructed to bridge the nerve stump by providing an inducive microenvironment for the regeneration of injured peripheral nerves.A viable NC is deemed to be closely associated with the orientation degree and bioactivity of the electrospun fibers.The aim of this thesis research is to fabricate highly aligned ultrafine fibers of PLLA containing graphene(Gr)by a novel stable jet electrospinning(SJES)method for the construction of NCs.Gr has good electrical conductivity and biocompatibility,and has been reported to induce differentiation of neural stem cells into neurons.So the introduction of Gr into the highly-aligned PLLA ultrafine fibers will likely increase the effectiveness of neural regeneration.First of all,after optimizing ultrasonic time for Gr dispersion highly aligned ultrafine fibers of Gr/PLLA composites with varied Gr ratios of 0,0.1,0.5,and 1% were prepared by the SJES method.In the meantime,semi-oriented PLLA fibers used as control were fabricated by CES method.The morphology and orientation degree of thus produced Gr/PLLA fibrous mats were examined by scanning electron microscope(SEM)and fast Fourier transform(FFT).Composition and Gr dispersion in PLLA fibers were examined by transmission electron microscope(TEM)and Raman spectroscopy.And thermal and mechanical properties were characterized by means of DSC,TGA and tensile tests.The results indicate that all the fibers have almost the same diameter around 1.50 ?m and the fibers made by SJES show better alignment than that by CES.The mechanical properties increase first and then decrease with increasing the Gr contents.The fibers containing Gr also have better thermal stability due to excellent thermal stability and high thermal conductivity of Gr.Introduction of a tiny amount of Gr(e.g.,0.1%)enhanced remarkably the thermal property of the Gr/PLLA fiber mats,leading to at least 20 ?C increment in decomposition temperature to reach the level of 50% weight loss.Biological properties of the aligned Gr/PLLA fiber scaffolds were firstly evaluated by performing assays relating to adhesion,proliferation and glial fibrillary acidic protein(GFAP)expression of the Schwann cells(SCs).Furthermore,effect of the aligned Gr/PLLA fibers on the differentiation of PC12 cells was also examined.The results indicated that compared with the control tissue culture plate(TCP)and semi-oriented fiber counterpart,all the highly aligned Gr/PLLA fiber scaffolds from SJES supported better cell adhesion,especially after cell-seeding for 8 h.Both the fiber orientation degree and the incorporated Gr content significantly affected the cell elongation,and increasing the Gr content gave rise to increased cell elongation percentage,demonstrating the promotional role of Gr introduction.All the fiber scaffolds could promote SCs proliferation with no significant difference between groups which indicates the fibrous scaffolds appropriate cytocompatibility.Meanwhile,examination on the SCs morphology indicated that highly-aligned Gr/PLLA fibers promoted better SCs elongation and orientation.After 4 days of culture,SCs continued to express GFAP with remarkable increases on the groups of 0.1% Gr/PLLA and 0.5% Gr/PLLA fibers,while the other groups were similar with TCP.This indicated that contact guidance and Gr introduction had a synergistic effect on function expression of SCs.The morphology and axon number result of PC12 cells showed that the ultrafine composite fiber containing Gr could promote cell differentiation better.The results collectively demonstrated that Gr incorporation and higher fiber orientation degree have synergistic effects on SCs elongation,proliferation and PC12 cells differentiation.The nerve conduits(inner diameter = 2 mm)with good axial fiber orientation degree and pressure resistance were successfully fabricated from these highly aligned Gr/PLLA fibers by reeling-heating method.The morphology and fiber diameter of nerve conduits were observed by SEM.It was found that the fiber morphology was as well as before with no obvious alteration in fiber diameters.Cell proliferation results of the SCs within the tubular NCs by CCK-8 assay indicated that there was no significant difference between different groups,which was consistent with the results on planar fibious scaffolds.The morphology of SCs in the middle part of the inner wall of conduits illustrated that the SCs grew entirely along the fiber direction of the well-aligned nerve conduits.These results demonstrate that 3D NCs have good cytocompatibility and play a guiding role in cell growth and thus enable to perform further study in future on the efficacy of Gr/PLLA conduits in inducing nerve regeneration in vivo.In summary,highly-aligned Gr/PLLA fibers were successfully fabricated by SJES.The composite fibers had good physical and chemical properties and biological compatibility.A combination of high degree of fiber orientation and Gr incorporation contributed to synergistic effects on elongation,orientation,proliferation,GFAP expression of SCs and the differentiation of PC12 cells.The nerve conduits made by reeling-heating method have good axial fiber orientation degree and pressure resistance,and could promote SCs proliferation and orientated growth.All the results suggest that Gr/PLLA composite scaffolds hold great potential for applications in neural tissue engineering.