| With the development of regenerative medicine technology,transplantation of neural stem cells(NSCs)offers a feasible strategy for the clinic therapy of central nervous system disease,such as spinal cord injury(SCI).Whereas,the low survival rate,poor retention,and uncontrollable differentiation of the transplanted NSCs greatly restrict their application in SCI repair.Fortunately,neural tissue engineering scaffolds,as carriers of the transplanted stem cells,not only effectively deliver stem cells to the lesion area of injured spinal cord,but also provide a viable solution to address the aforementioned issues.Recently,the development of three-dimensionally(3D)bioprinting technology has offered an attractive approach for the precise fabrication of the neural tissue engineering scaffolds,attributed to its advantages in customized manufacture.Lacking of cell signal transmission path between stem cells and scaffold matrixes,traditional 3D printed scaffolds,such as collagen,chitosan,hyaluronic acid,are often difficult to regulate the fate of stem cells,nor suitable for sophisticated structures of nerve tissues,thereby resulting in a confined application of 3D bioprinting technology in nerve tissue engineering.As a consequence of the electrophysiological activities in central nervous system,it has been previously proved that the bioelectrical signal propagation between cells and scaffolds is of benefit to neuronal maturity and nerve tissue development.Therefore,it is a key to an improved therapeutic effect of transplanted NSCs in SCI repair,that electroconductive hydrogel(ECH)scaffolds with high conductivity and spinal cord-inspired structure are chosen as vehicles of NSCs.In this dissertation,the main efforts have been devoted to regulate the controllable neuronal differentiation of the transplanted NSCs,and precisely construct 3D biomimetic neural scaffolds for SCI repair.To realize this goal,an NSC-laden 3D ECH scaffold has been fabricated using 3D bioprinting technology,in which an enhanced neuronal differentiation efficiency is gained for the encapsulated NSCs,thus its applications in NSC transplantation and SCI repair has been explored.The following three main parts were included in this dissertation.In the first part of this dissertation,by the rational design of ECH formula,a molded ECH scaffold containing NSCs was prepared,followed by the investigation of NSC behavior in the ECH scaffolds.In this section,a novel conductive polymer PEDOT:CSMA,TA with well water solubility and conductivity,was firstly synthesized via in situ template-mediated polymerization,and was integrated with gelatin methacrylate/polyethylene glycol diacrylate precursor solution(GelMA/PEGDA),followed by photo-initiated crosslinking,obtaining ECH scaffolds with well conductivity(4×10-3 mS/cm)and suitable mechanical strength(0.5 kPa).The in vitro experiments indicated that the encapsulated NSCs in the molded ECH scaffolds not only had normal cell activity and proliferation behavior,but also extended richer filopodia,in comparison with those in the pure GelMA/PEGDA hydrogel scaffolds.More importantly,both the increase of neuronal differentiation and the inhibition of astrocytic production were meanwhile observed for the NSCs encapsulated in the ECH scaffolds,attributing to the introduction of PEDOT:CSMA.TA,successfully testifying an induced effect of the ECH scaffolds on NSC differentiation.Therefore,this work lays a basis on the application of the ECH scaffolds in NSC transplantation and nerve tissue repair.Aiming to achieve precise fabrication of 3D neural scaffolds with biomimetic structure,in the second part of this dissertation,an original concept of a dynamicviscoelastic bioink was employed to explore physical gel-based and complementary polymer network-based bioinks with optimized formulas,respectively.According to rheological analysis of the dynamic-viscoelastic bioinks,and the structural parameters and cell behaviors of 3D-printed hydrogel scaffolds,the bioprinting process of these dynamic-viscoelastic bioinks has been successfully preformed with bioink formula screening,as evidenced by shape fidelity,printing resolution,physiological stability,mechanical stability and cytocompatibility of 3D-printed scaffolds.Furthermore,the application of the dynamic-viscoelastic bioinks in neural tissue engineering has been explored via neuronal-like cell-laden bioprinting,and the immunofluorescent staining results showed that the cells in the 3D-printed scaffolds presented the neuronal morphology with neurite outgrowth.Consequently,the study of the second part not only provides a methodological instruction into 3D bioprinting of ECH-based neural scaffolds,but also extends the application of 3D bioprinting technology in neural tissue engineering.As an integral trial to fabricate a biomimetic neural tissue transplant used in NSC delivery and SCI repair,in the final part of this dissertation,inspired by nerve fiber bundle of spinal cord,an optimized physical gel-based composite bioink,containing PEDOT:CSMA,TA,GelMA/PEGDA precursor solution,and NSCs,was gained to build an NSC-laden ECH composite scaffold with an axial-stacking structure,via 3D bioprinting technology,for regulating NSC proliferation and differentiation in SCI repair.The in vitro tests demonstrated that the NSCs in the ECH composite scaffold not only survived with high cell viability(nearly 100%)and normal adherence,but also tended to differentiate into neurons with extended neurites.On the other hand,an enhanced therapeutic efficiency in SCI repair was observed for in vivo transplantation of the NSC-laden ECH composite scaffold,probably ascribable to a synergistic effect of the ECH scaffold in controllable neuronal regeneration,inhibited glial scar formation,improved axonal growth and neural tissue development.As a feasible therapeutic strategy,thus-formed ECH scaffold is of vital significance and applied value in the further basic research and clinic translation of neural tissue engineering and regenerative medicine. |
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