Electrospinning Plla/Graphene Nanofibers For Neural Tissue Engineering

In tissue engineering, repairing and regeneration of injured nervous tissues still remains to be one of the most intensively investigated topics. But recent nanotechnological advancements in fabrication of nano-and/or micro-fibrous scaffolds that mimic the natural extracellular matrix (ECM) via electrospinning, could offer an innovative strategy and approach to achieve improved efficacy in repairing and regeneration of nervous system impairment. Meanwhile, graphene, a new2-dimensional nanomaterial consisting of hexagonal hive lattice with a single atomic layer of sp2-bonded carbon atoms, has been demonstrated to promote neural cell proliferation and differentiation of neural stem cells (NSCs). However, research in incorporating graphene into electrospun biomimetic nanofibers for nerve tissue scaffolding is rarely reported. The objectives of this study are therefore to fabricate graphene-incorporated poly(L-lactic acid)(PLLA) composite nanofibers (i.e., PLLA/Graphene) by electrospinning, and to explore the cytocompatibility as well as capacity of the nanofibrous PLLA/Graphene scaffolds in regulating neural differentiation of NSCs in vitro, from which the potential of using the PLLA/Graphene composite nanofibers for neural tissue engineering could be identified.First of all, parameters for ultrasound treatment were optimized to better disperse the graphene powder into hexafluoroisopropanol (HFIP), from which composite nanofibers of PLLA/Graphene were fabricated by electrospinning to attain homogeneous dispersion of graphene nanosheets within PLLA nanofiber matrix as well. Morphology and dispersion effect of graphene nanosheets in PLLA nanofibers were characterized by SEM, TEM and Raman spectroscopy. Thermal and mechanical properties as well as wettability of the nanofibrous PLLA/Graphene scaffolds were examined by DSC (and TG), tensile tests and water contact angle measurements, respectively. It is found that the graphene could be well dispersed within PLLA nanofibers. Diameters of the electrospun PLLA/Graphene nanofibers show a decreasing tendency initially with increasing the graphene content from0to1wt%(the minimal fineness is in the range of0.50±0.19μm), thereafter increase slightly when the graphene loading was increased to2wt%. Coincidently, both the ultimate tensile strength and crystallinity of the electrospun PLLA/Graphene reached maxima at1wt%loading of graphene, that is, a50%increase in tensile strength (compared to electrospun pristine PLLA) and maximal crystallinity up to55%. In addition, the electrospun PLLA/Graphene also exhibited improved thermal stability.Cytocompatibility of the electrospun PLLA/Graphene nanofibers was evaluated by detecting cell viabilities of a glial cell line, Schwann cells (SCs), which were in vitro cultured on the composite nanofibrous substrates. After culturing the SCs-scaffold constructs for1,4, and7days, MTT assay and SEM observation were performed to determine the cell proliferation and morphology, respectively. There appeared a sharp enhancement in the SCs’viability based on the quantitative MTT data. SEM results showed that the cells adhered and spread well to the graphene-incorporated nanofibrous PLLA scaffolds, with visible pseudopodia protruded. Additionally, cytocompatibility evaluation using myocardial cells (derived from SD rats) as another electrical signal sensitive cell source for seeding on the nanofibrous PLLA/Graphene scaffolds demonstrated similar outcomes. These results prove that the newly developed PLLA/Graphene nanofibers are of good cytocompatibility with undetectable cytotoxicity.To investigate the biological functions of the electrospun PLLA/Graphene nanofibers, we evaluated the differentiation ability of the NSCs on the nanofibrous scaffolds by SEM and immunocytochemical staining. After culturing the NSCs for14days, SEM data showed that the NSCs adhered well to the nanofibrous PLLA/Graphene scaffolds and extended to the surrounding area. Immunocytechemical staining further indicated that PLLA/graphene nanofibers support enhanced NSC differentiation towards neurons, astrocytes and oligodendrocyte lineages in vitro by giving rise to nervous system relevant protein expressions. There was a strong positive expression of GFAP protein marker with the nanofibrous PLLA scaffolds containing graphene. These results suggest that the NSCs could adhere well to the nanofibrous PLLA/Graphene scaffolds and the incorporated graphene played an important role in directing the NSCs towards neural differentiation.In summary, for the first time this study demonstrated the successful incorporation of graphene nanosheets into the PLLA nanofibers by electrospinning. Compared to electrospun PLLA nanofibers free of graphene, the nanofibrous PLLA/graphene scaffolds possess enhanced physical and chemical properties. And also, apart from good cytocompatibility (with respect to those cells sensitive to electrical signals, e.g., Schwann cells and myocardial cells), the nanofibrous PLLA/graphene scaffolds were also found to promote differentiation of NSCs to neurons, astrocytes, and oligodendrocyte lineages in vitro. All the results obtained in this study suggest the great potential of using the electrospun PLLA/graphene composite nanofibers for applications in basic and clinical research of neural tissue engineering, worthy of further researches in future.