Controllable Construction Of Electrospun 3D Aligned Nanofibrous Scaffolds And Their Application In Spinal Cord Regeneration

The repair of spinal cord injury（SCI）is a burning medical problem all over the world.At present,surgical decompression and methylprednisolone administration are mainly employed to treat SCI in clinics.Unfortunately,these strategies can’t promote the healing of SCI,which can only prevent the injury from getting worse.The long-term follow-up treatment effect is pretty poor for SCI patients.Compared with the existing therapies,tissue-engineered scaffolds exhibited potential advantages.The tissue-engineered scaffolds can be filled into the SCI gap and facilitate the neuronal tissue regeneration and functional recovery.In recent years,electrospinning has drawn intense attraction among numerous scaffold-forming techniques.However,the currently-fabricated electrospun nanofibers are mainly collected in the form of two-dimensional（2D）mats.The nanofiber mats exhibited some merits,like small fiber diameter,high specific surface area and excellent extracellular matrix（ECM）fibril-mimicking characteristics,but their dense structure and small pore size commonly inhibit the cell migration and infiltration into the inner parts of 2D electrospun mat scaffolds.Worse still,2D electrtospun scaffolds can’t resemble the three-dimensional（3D）structure,mechanics and properties of native spinal cord tissues,and lack of advanced3D physical guidance for the regeneration of damaged spinal cord.In this thesis,an innovative technical routine combining electrospinning with subsequent gas-forming treatment was designed and developed to generate polycaprolactone（PCL）/poly（p-dioxanone）（PPDO）three-dimensional（3D）nanofibrous sponges（NSs）.The electrospinning process and gas-forming process were systematically optimized.The finally-obtained 3D PCL/PPDO NSs were expected to possess a laminated structure with ECM-mimicking aligned nanotopography,controllable hierarchical structure,high porosity and hydrophilicity,thereby providing an instructive microenvironment to induce the regeneration of the damaged spinal cord.Moreover,exogenous neural stem cells（NSCs）harvested from foetal rats were introduced into the above-mentioned 3D PCL/PPDO NS system,which were expected to offer synergistic effects on promoting the regeneration and functional recovery of large-scale SCI.（1）The effects of PCL/PPDO mass ratios on the morphology,surface properties,and degradation rate,as well as mechanical and biological performances of 2D electrospun nanofibrous mats（NMs）were systematically investigated.The results showed that the mixture of PCL and PPDO effectively made up for the deficiency of single polymer,and the properties of as-prepared NMs could be controllably regulated by changing the PCL/PPDO mass ratio.All 2D PCL/PPDO NMs presented highly oriented fibrous structure.Specifically,the water contact angle of pure PCL NMs was 117.5±1.6°,the longitudinal（along the fiber orientation direction）and transverse（perpendicular to fiber orientation direction）ultimate strength were 41.2±2.3 MPa and 4.2±0.1 MPa respectively.With the addition of PPDO component,the surface hydrophilicity of 2D PCL/PPDO NMs gradually increased,but the ultimate strength gradually decreased.Both 4:1 and 1:1 PCL/PPDO NMs could significantly promote adhesion and proliferation of human adipose derived mesenchymal stem cells（h ADMSCs）in comparison with the other NMs with different PCL/PPDO mass ratios.（2）The influences of sodium borohydride（Na BH₄）concentration,gas-forming time,and Pluronic F127 addition content for the preparation of 2D PCL/PPDO nanofibrous mats on expansion height,porosity and water absorption of finally-generated 3D PCL/PPDO NSs were explored.The results showed that all the effects were positive.The expansion height of 3D NSs was up to 13.70±2.71 mm,which increased by 105.4 times compared with the thickness of 2D PCL/PPDO NMs.The maximum porosity and water absorption of 3D NSs could reach to 99.16±0.20%and 41.90±3.29 g/g,respectively,which were 1.3times and 4.6 times than those of 2D NMs,respectively.Based on the defect size of rat SCI models,3D PCL/PPDO NSs prepared from 2 M Na BH₄ and 20 min of expansion were chosen as the potential scaffolds for animal experiments,and the micro-morphology,surface hydrophilicity and mechanical properties of as-chosen 3D NS scaffolds were determined and analyzed in detail.The 3D NSs exhibited highly-aligned nanofibrous and controllable 3D layered structure,the porosity（98.12±0.75%）was significantly higher than that of 2D NM control group（75.46±3.21%）.The hydrophilicity was also demonstrated to be obviously higher than that of the 2D NM control group.More detailly,3D NSs could completely absorb water droplets within 2 s,presenting excellent wettability.More importantly,compared with the 2D NM control group,3D NSs exhibited more reasonable mechanics.The Young’s modulus of 2D NMs was up to 89.34±5.09 MPa,while that of 3D NSs was only 0.61±0.12 MPa,which was close to normal spinal cord tissue（0.2-0.6 MPa）and more suitable for SCI treatment.（3）How the 3D PCL/PPDO NSs affected the cell behaviors like viability,proliferation and differentiation of neural stem cells（NSCs）were determined by cell culture experiments.In addition,the effects of 3D NSs carried with and without exogenous NSCs on the tissue regeneration and functional recovery of damaged spinal cord tissues in rats were further characterized.The in vitro cell culture experiments showed that 3D NSs possessed excellent biocompatibility to NSCs.The 3D NSs with excellent ECM fibril-mimicking structure,highly-aligned nanofibrous and controllable 3D layered structure could significantly promote the migration and infiltration of NSCs into the internal parts of 3D NSs.The In vivo animal experiments indicated that the 3D NSs could significantly promote the migration and infiltration of autologous neuronal cells to further induce the axon outgrowth and restore the continuity of damaged spinal cord in the SCI defect area.Moreover,the exogenous NSCs were demonstrated to exhibit high survival rate in rats,and could further differentiate into mature neurons within the 3D NSs.The combination of 3D nanofibrous sponge and exogenous NSCs were found to possess synergetic effects on neuronal regeneration and functional recovery in rat SCI models.It should be noticed that both the microstructure of regenerated tissues and functional recovery degree in exogenous NSCs-loaded 3D NSs group were closer to the sham control group.Overall,the present study indicates that the as-fabricated 3D NSs can effectively regulate the fate of NSCs,and an advanced combination of 3D NS design and transplanted NSCs invites applications as an ideal tissue-engineered scaffold for SCI repair.