| Objective: Peripheral nerve injury(PNI)is a common neurosurgical disease,mainly manifesting as motor dysfunction,sensory dysfunction,and neurotrophic changes.After peripheral nerve trauma,if the defect is large and cannot be reunited even after the adequate freeing of the two severed ends,an autologous nerve graft is usually used.However,disadvantages include a lack of donor source,secondary damage to the donor site,or loss of nerve sensory function at the donor site.Other treatment modalities such as physical therapy,drugs,and electrical stimulation are ineffective.Biomaterial-based nerve guidance conduits(NGCs)have emerged as a promising peripheral nerve injury regeneration alternative.Bridging the injury site with a nerve conduit can provide a suitable microenvironment for axonal regeneration,improve the efficiency of nerve regeneration,and restore damaged motor and sensory functions.However,most of the NGCs currently used in clinical practice are hollow structures with disadvantages such as easy collapse and poor permeability.This study reported a method to prepare filled NGCs with three-dimensional structures based on galectin-1 deposited on radially aligned nanofiber yarns with a unidirectional linear gradient by controlling the electrostatic field distribution during electrospray process,which provides a new idea for clinical treatment of peripheral nerve injury.Methods:1.Preparation of polycaprolactone(PCL)Nanofiber yarns(NY).The polycaprolactone(PCL)was dissolved in Hexafluoroisopropanol(HFIP),and the concentration of PCL was 12%(w/v).Then the nanofiber yarns were obtained continuously and stably using electrostatic spinning equipment at a rate of 0.020 ml/min at a high pressure of 9.8 k V.2.Determination of galectin-1 concentration.Schwann cells(SCs)and PC12 cells(rat adrenal pheochromocytoma cells)were cultured with galectin-1 at different concentrations(0 ng/m L,5 ng/m L,20 ng/m L,and 50 ng/m L).The effects of different concentrations of galectin-1 on the proliferation and viability of these two cells were detected by the CCK-8method.The effects of different concentrations of galectin-1 on the morphology of SCs and the growth of PC12 axons were analyzed by cellular immunofluorescence staining and axonal staining,respectively.The relative optimum concentration of galectin-1 was determined by the above methods.3.Formation of galectin-1 linear gradient coating on PCL nanofiber yarns.A galectin-1unidirectional linear gradient coating was formed on PCL nanofiber yarns by controlling the electrostatic field distribution during electrostatic spraying.4.The pure nanofiber yarn membrane,uniform galectin-1 coating nanofiber yarn membrane,and unidirectional linear gradient galectin-1 coating nanofiber yarn membrane were constructed.The viability,morphology,and migration of SCs and neural stem cells(NSCs)on the above three nanofiber membranes were investigated.Blank slides were used as the control group to provide a basis for the subsequent preparation of NGCs with a three-dimensional structure.5.Preparation of the inner and outer walls of NGCs.Three structures of PCL nanofiber membranes with the aligned,yarn,and conjugated yarn were prepared by electrostatic spinning technique.The proliferation,morphology,and cytoskeleton growth direction of SCs on these three PCL nanofiber membranes were studied by the CCK-8 method.As well as the microstructural characteristics of the three PCL nanofiber membranes were observed by emission scanning electron microscopy to determine the material structure of the inner wall of the NGCs.A common single-nozzle random electrostatic spinning method prepared the outer wall of NGCs with a random topology.6.Preparation of hollow nerve conduits.The inner wall of the NGCs by conjugation technique,and the outer wall of the NGCs with random topology prepared by single-nozzle random electrostatic spinning technique,the resulting bilayer nanofiber membrane was rolled into a tube shape.The hollow NGCs were successfully prepared by welding the overlapping nanofiber membranes using a laser emitter.7.Preparation of three types of filled PCL nanofiber yarns nerve conduits.PCL Nanofiber yarns-encapsulated PCL NGCs(NY-PCL NGCs),galectin-1 coating,and PCL nanofiber yarns-encapsulated PCL NGCs(GC-NY-PCL NGCs).The Hyaluronic acid Methacryloyl(HAMA)filled galectin-1 coating and PCL nanofiber yarns-encapsulated PCL NGCs(HA-GC-NY-PCL NGCs)were prepared based on the hollow nerve conduits and galectin-1gradient coating described above.And the microstructural features of NGCs were observed by emission scanning electron microscopy.The cross and longitudinal sections of NGC-cell composites were obtained by frozen section.The samples were stained with immunofluorescence to observe the migration and distribution of SCs and NSCs in the above NGCs.Results:1.The PCL nanofiber yarns were successfully prepared.The average yarn diameter is 192μm,and the distribution is highly normal.2.CCK-8 results showed that SCs proliferated fastest in the 50 ng/m L group,significantly different from all other groups(P < 0.05).The axon length and viability of PC12 cells on day 4 were counted and analyzed,and the results showed that the axon length and cell viability of the 50 ng/m L group were higher than those of the other groups(P < 0.05).3.Fluorescence microscopy showed that the PCL nanofiber yarn membrane produced a unidirectional linear gradient of gelatin/ galectin-1 loaded with rhodamine B.4.Migration experiments of SCs and NSCs revealed an increase in the number of cells in the entire migration region of the galectin-1 coating group with the unidirectional linear gradient.The total number of migrating cells was significantly higher in the gradient group than in all other groups(P < 0.05).The viability results on day 3 showed that the gradient group had the highest survival rate of NSCs and SCs.5.SEM images indicated that PCL nanofiber membranes with three structures of the aligned yarn and the conjugate yarn were highly oriented.The SCs cultured on aligned and conjugate yarn nanofiber membranes adhered and grew along the ordered axial orientation.CCK-8 results showed that the proliferation of SCs on conjugate nanofiber membranes was better than all other groups(P < 0.05).6.The four kinds of PCL NGCs were successfully prepared.The diameter was about 2 mm,and tens of nanofiber yarns were inside the lumen.In the HA-GC-NY-PCL NGCs,the HAMA was filled between the nanofiber yarns and conduit,effectively supporting the nanofiber yarn and maintaining the structure of NGCs.7.It was observed from the cross-sectional immunofluorescence images that there were more SCs and NSCs between adjacent nanofiber yarns of HA-GC-NY-PCL NGCs.Observing the longitudinal section immunofluorescence images,the number of adhesions and migration of SCs and NSCs within the lumen of the HA-GC-NY-PCL NGCs were significantly higher than in all other groups.Conclusion:1.The PCL NGCs were successfully prepared by electrospinning technology,in which the inner wall was a conjugate structure,and the outer wall was a random topological structure.2.The nanofiber yarns with a unidirectional linear gradient of galectin-1 coating were successfully fabricated by controlling the electrostatic field distribution during the electrospray process.3.Galectin-1 unidirectional linear gradient coating can significantly promote the biological viability of SCs and NSCs,which is reflected in improving cell viability and migration.4.The designed three-dimensional structure of HA-GC-NY-PCL NGCs has better guidance for migrating SCs and NSCs. |
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