The Development Of Modular Bioink Based On Hydrogel Microspheres For 3D Bioprinted Nerve Tissue Engieering Scaffold Applications

Research Background:After peripheral nerve injury,nerve regeneration is slow and incomplete,which negatively affect the functional recovery of patients,and take a risk to endure secondary disabilities or other secondary injuries.Peripheral nerve repair is always without complete functional recovery,and an autologous nerve graft for nerve gap remained a critical challenge in the clinical treatment due to the limitation of the spontaneous graft.Recently,nerve tissue engineering has been encouraged to fabricate peripheral nerve scaffolds and alternative nerve grafts in the field of nerve regeneration.In nerve tissue engineering,the regulation of neuron extension plays an important role.At present,electrospinning,micropattern,and self-assembly techniques have been used to prepare nerve scaffolds,mimicking the structure of native nerve tissue and direct the growth and extension of nerve.In the design of peripheral nerve tissue scaffolds,the integration of micro and macro-environments plays an important role.Therefore,developing multiscale scaffolds to enhance the maturation and elongation of 3D neurons remains challenging.Objective:In this study,microfluidic technology was used to prepare hydrogel microsphere modular bioink,and 3D printing technology was used to prepare multiscale composite scaffold,which integrated 3D micro and macro-environment to simulate natural neural tissue.To explore the preparation of a good neuronal microenvironment based on methacrylic anhydride gelatin/chitosan composite microspheres(GelMA/Chitosan Microspheres,GC-MS),PC 12 cells and neurite outgrowth were promoted.Based on microspheres and hydrogels as modular bio-inks,3D multiscale composite scaffolds were bioprinted.The co-culture system of PC 12 cells and RSC96 cells in the bioprinted multi scale scaffolds was studied to evaluate neurite outgrowth and Schwann cell proliferation.Methods:1.The microfluidic method was used to prepare GC-MS,a microscope was used to analyze the size and distribution of GC-MS,and scanning electron microscope was used to observe the surface microstructure of GC-MS.2.The fluorescence-labeled NGF was loaded into GC-MS,the changes of the swelling degree of the microspheres and the fluorescence intensity of NGF were analyzed,and the sustained releasing of NGF was studied.3.PC12 cells were seeded on GC-MS,Live/Dead vitality detection kit was used to detect cell viability,Alamar Blue kit was used to study cell proliferation,and cell morphology and neurite outgrowth was analyzed by cytoskeleton fluorescence staining and scanning electron microscope4.The microspheres and GelMA hydrogel was used to fabricate a modular composite.bioink and the composite scaffolds were prepared by bioprinting technology.The structure of composite scaffold was analyzed by a fluorescence microscope,and the application of the cell co-culture system in nerve repair was evaluated.Results:1.A series of multifunctional GC-MS were prepared by the microfluidic method,the hydrogel microsphere with diameters ranging from 54±5 ?m to 350±21 ?m were formed after injecting different flow rates aqueous phase solution(10,15,20?L/min)and oil phase solution(75,225,300,450,600 ?L/min).2.When PC 12 cells were seeded on GC-MS,it was found that GC-MS had excellent biocompatibility.At the same time,GC-MS loaded with NGF encouraged the proliferation and axonal outgrowth of PC 12 cells.3.A multiscale composite scaffold composed of GC-MS and GelMA hydrogel was successfully printed to mimic the multiscale network structure of native nerve tissue.After the encapsulated PC 12 cells were cocultured with RSC96 cells,the result of fluorescence staining showed the extension of the cells in the scaffold.Conclusions:these results suggest that GC-MS+NGF as a cell carrier can enhance the neurite outgrowth and extension,and hydrogel will mimic the epineurium layer to provide a 3D macroenvironment for Schwann cells proliferation and nerve cell organization,which indicates that multiscale core-shell biomimetic composite scaffold is promising for the great applications in peripheral nerve tissue engineering.