The Preparation Of Three-dimensional Conductive Scaffolds And Their Effect On The Proliferation And Differentiation Of Neural Stem Cells In Vitro

Neural tissue engineering provides a viable approach to the treatment of neurological diseases and its essence is the effective integration of a variety of microenvironmental factors conducive to nerve regeneration for achieving nerve repair and regeneration.Seed cells,scaffolds,and growth information are the three key elements that make up tissue engineering.As a kind of cells with self-renewal and multi-directional differentiation potential,neural stem cells(NSCs)are considered as ideal seed cells for neural tissue engineering.Recently,studies have found that conducting polymers(CPs)as a new type of "smart" material can modulate the behavior of NSCs individually or in combination with electrical stimulation(ES).However,the development of tissue engineering scaffolds based on CPs is still immature,such as inefficient preparation method,low conductivity of scaffold and single structure of scaffold,which makes it difficult to meet the needs of tissue engineering applications.Considering that poly(3,4-ethylenedioxythiophene)(PEDOT)is superior to other conductive polymers in terms of photoelectric properties and environmental stability,novel three-dimensional conductive scaffolds based on PEDOT in this study were developed to regulate the proliferation and directional differentiation of NSCs in combination with electrical stimulation,which may provide a possible pathway for nerve repair and regeneration.In this paper,NSCs derived from fetal rat hippocampus were successfully isolated and cultured.The cell growth curves under different inoculation densities showed that the optimal inoculation density of NSCs cultured in vitro was 1 × 105-2 × 105 cells/mL,and the suitable passage time was 5-7 days.Through the analysis of related proteins and genes,the cultured NSCs were identified as self-renewal and multi-directional differentiation potential.NSCs express their specific markers(Nestin＋/Sox2＋/CD133＋/CD34-/CD45-)and can be differentiated into neurons(β tubulin-Ⅲ＋),astrocytes(GFAP＋)and oligodendrocytes(04＋).Therefore,the cultured cells possess the biological characteristics of NSCs and can be used for subsequent experimental research.Because PEDOT has excellent photoelectric properties,environmental stability and good biocompatibility,it has been developed for the construction of conductive tissue scaffolds for nerve tissue engineering.In this study,PEDOT-HA/Cs/Gel and PEDOT/Cs/Gel three-dimensional conductive scaffolds were prepared and their physicochemical properties and biocompatibility were evaluated.We selected hyaluronic acid(HA)as a dopant,PEDOT-HA conductive nanoparticles with a diameter of about 200-300 nm were prepared by chemical polymerization.Then,PEDOT-HA was successfully introduced into chitosan and gelatin macromolecules by chemical cross-linking to obtain PEDOT-HA/Cs/Gel conductive scaffolds.In addition,considering the water absorption of chitosan and gelatin,and the characteristic of the monomer 3,4-ethylenedioxythiophene(EDOT)dissolved in the organic phase,PEDOT/Cs/Gel conductive scaffolds were prepared by in situ interfacial polymerization of PEDOT conductive nano-particles with a diameter of about 50 nm on the wall surface of Cs/Gel scaffold.Physicochemical characterization of the conductive scaffolds showed that the introduction of PEDOT reduced the water absorption and in vitro degradation rate of the scaffold,but increased the surface roughness,electrical conductivity and compressive modulus of the scaffold.The conductivity of PEDOT/Cs/Gel scaffolds was significantly higher than that of PEDOT-HA/Cs/Gel scaffolds.Biocompatibility results showed that both PEDOT-HA/Cs/Gel and PEDOT/Cs/Gel conductive scaffolds were conducive to the adhesion of PC12 cells in scaffolds.Particularly,8%PEDOT-HA/Cs/Gel scaffolds maintained good cell proliferative activity,while 2PEDOT/Cs/Gel scaffolds significantly enhanced cell proliferation.In order to study the effect of conductive scaffolds prepared on the behavior of NSCs,8%PEDOT-HA/Cs/Gel and 2PEDOT/Cs/Gel scaffolds were selected to culture NSCs.Cell adhesion,metabolism,proliferation and differentiation of NSCs in conductive scaffolds were investigated.Compared with Cs/Gel scaffolds,PEDOT-HA/Cs/Gel and PEDOT/Cs/Gel scaffolds significantly promoted the adhesion of NSCs in scaffolds.NSCs in the conductive scaffolds were cultured continuously for 14 days under the proliferation condition.It was found that the conductive scaffolds were conducive to the proliferation of NSCs,and increased the protein and gene expression of Nestin,Sox2 and Ki67.The results of cell metabolism also showed that NSCs could maintain a high rate of cell proliferation in the conductive scaffolds.Moreover,NSCs were expanded in proliferation medium for 4 days,then differentiated with differentiation medium for 6 days.Cell immunofluorescence staining and RT-qPCR results showed that PEDOT-HA/Cs/Gel and PEDOT/Cs/Gel scaffolds promoted the differentiation of NSCs into neurons(β tubulin-Ⅲ+)and astrocytes(GFAP+),and upregulated the expression of related proteins and genes.In addition,there was no significant difference between PEDOT-HA/Cs/Gel and PEDOT/Cs/Gel scaffolds in influencing the adhesion,metabolism,proliferation and differentiation of NSCs.Finally,we further investigated the effect of conductive scaffold on the proliferation and differentiation of NSCs in combination with electrical stimulation.NSCs in the conductive scaffolds were electrically stimulated at different voltage intensities(0 mV,25 mV,50 mV,100 mV and 200 mV)for 7 days(4 h/day).The highest cell viability was achieved at 50 mV and 100 mV in the PEDOT/Cs/Gel and PEDOT-HA/Cs/Gel scaffolds,respectively.The results of protein and gene expression showed that electrical stimulation not only promoted the proliferation of NSCs in the conductive scaffold,but also facilitated the differentiation of NSCs into neurons.Additionally,the role of conductive scaffolds in combination with electrical stimulation on the differentiation of NSCs was studied in the presence of ion channel blockers.It was found that electrical stimulation significantly increased the differentiation of NSCs into neurons and enhanced the intracellular Ca2+ fluorescence intensity of differentiated cells.Conductive scaffolds combining with electrical stimulation may be through the regulation of cell membrane surface Ca2+,Na+,K+ and Cl-voltage-gated ion channels to act NSCs differentiation,Ca2+ and Na+ channels probably play a major role.In summary,PEDOT-HA/Cs/Gel and PEDOT/Cs/Gel three-dimensional conductive scaffolds have good electrical activity and biocompatibility.Conductive scaffolds could not only promote NSC adhesion and proliferation,but also enhance the differentiation of NSCs into neurons and astrocytes.Importantly,conductive scaffolds can be combined with electrical stimulation to further enhance the differentiation of NSCs into neurons.It is expected that these electroactive scaffolds will be used as neural tissue engineering scaffold to regulate the directional differentiation of NSCs in combination with electrical stimulation.