The Study Of Low Temperature Extrusion 3D Printing BDNF/collagen/chitosan Scaffold To Repair Spinal Cord Injury

The Objective:incidence of spinal cord injury(SCI)is increasing every year,and effective treatment has always been a difficult problem for the medical field.Recent studies have shown that 3D printing scaffolds integrated with growth factors can guide the growth of neurites and promote axonal regeneration at the injury site.However,conventional 3D printing can reduce the biological activity of growth factors by using heat,organic solvents or cross-linking agents,resulting in the inability to incorporate growth factors for controlled release.Low temperature 3D printing can incorporate growth factors into the scaffold and maintain the biological activity of growth factors during printing.Our aim was to develop a collagen/chitosan scaffolds integrated with brain-derived neurotrophic factor(3D-CC-BDNF)by low temperature extrusion 3D printing as a novel artificial controlled release system for the treatment of SCI.Methods:1.Preparation of the scaffold and determination of the mechanical properties of the scaffold.3D-CC-BDNF was prepared by low temperature extrusion 3D printing technology.The gravimetric method was used to measure the water absorption of the stent,and the volume method is used to measure the porosity.In addition,the Instron5865 mechanical tester was used to measure the compression modulus of the scaffold.To measure the stability of the scaffold,a differential scanning calorimetry(DSC)was performed with a synchronous differential thermal analyzer.The FTIR-ATR spectrum of the scaffold was analyzed using a FTIR spectrometer.2.Determination of BDNF release kinetics by scaffold.The amount of BDNF in the supernatant was analyzed at each time interval using an enzyme-linked immunosorbent assay(BDNF Immunoassay,R & DSystems?,USA)according to the manufacturer’s instructions.The cumulative amount of BDNF released and the amount of BDNF were then calculated.3.Cytocompatibility testing of scaffolds in vitro.Isolation and identification of neural stem cells(NSCs)or human umbilical cord mesenchymal stem cells(HUCMSCs).The pipettes were used to inoculate fourth-generation NSCs or HUCMSCs onto the washed scaffolds at a density of 1 × 106 / ml.The phase morphology and growth of the cells on the scaffold were observed under a phase contrast microscope,scanning electron microscope,HE staining,and fluorescence microscope.Cell adhesion was measured.The cell adhesion rate of NSCs was calculated as follows: Cell adhesion rate =(number of adherent cells / number of seeded cells)× 100%.CCK-8 was used to measures the viability of NSCs or HUCMSCs during co-culture.4.SCI modeling and stent transplantation.Adult female SD rats(220-250 g,n = 80)was selected.A 2 mm spinal cord segment was completely excised at the T10 level of the rat with small scissors to establish a T10 completely transected SCI model.Immediately after hemostasis,the scaffolds were transplanted into the gaps separately to bridge the space between the transected tissues.All rats were randomly divided into four groups: Sham group(only laminectomy without SCI,n=20),SCI group(SCI without transplantation,n=20),3D-CC+BDNF group(3D-CC+BDNF implanted into completely-transected gap,n=20),3D-CC-BDNF group(3D-CC-BDNF implanted into completely-transected gap,n = 20).5.Detection of spinal cord repair.Behavioral assessment(The Basso–Beattie–Bresnahan(BBB)tests and the inclined-grid climbing tests),electrophysiological studies,MRI,DTI,HE staining,Bielschowsky’s silver staining,immunofluorescence staining,the Biotinylated dextran amine(BDA)tracing and transmission electron microscopy(TEM)was performed to assess the degree of SCI repair.Results:1.Light microscope,fluorescence microscope,SEM and HE staining images showed that the 3D scaffold was porous.The water absorption of 3D-CC-BDNF was lower than that of 3D-C-BDNF and 3D-C+BDNF(P <0.05).The porosity of3D-CC-BDNF was higher than that of 3D-C-BDNF and 3D-C+BDNF(P <0.05).Compared with C+ BDNF((16.58 ± 4.70)Kpa),CC + BDNF((28.91 ± 5.31)Kpa)and 3D-C-BDNF((53.56 ± 6.01)Kpa),the elastic modulus of 3D-CC-BDNF was increased((64.16 ± 5.27)Kpa(P <0.05).DSC indicated that the Tm value of pure BDNF was 5 ° C lower than the Tm value of 3D-CC-BDNF.Infrared spectral data showed that 3D-CC + BDNF and 3D-CC-BDNF had suitable fat-soluble and water-soluble chemical bonds.The infrared spectrum absorption peak of the3D-CC-BDNF at 3441.587 cm-1 significantly exceeded that of the 3D-CC+BDNF.2.Compared with 3D-CC+BDNF,3D-CC-BDNF released more BDNF,the release process was more stable,and the release lasted longer.3.The results of CCK-8 showed that both 3D-CC+BDNF and 3D-CC-BDNF had cytocompatibility.Compared with the 3D-CC + BDNF group,the 3D-CC-BDNF group was more conducive to cell growth.4.At 1 day after SCI,all hind limbs of the SCI rats were paralyzed,while the hind limbs of the rats in the Sham group were normal.Over time,the results of behavioral assessment and electrophysiological studies indicated that compared with the3D-CC+BDNF group,the rats in the 3D-CC-BDNF group exhibited better lcomotor function recovery.At 8 weeks after modeling,the results of MRI and DTI indicated that 3D-CC-BDNF transplantation could promote the regeneration of nerve fiber tracts after SCI compared with 3D-CC+BDNF transplantation.At 8 weeks after modeling,the macroscopic histology of the spinal cord,HE staining and Bielschowsky’s silver staining and immunofluorescence staining showed that the implanted 3D-CC-BDNF filled the gap of the injury site,promoted the regeneration of nerve fibers and accelerated the establishment of synaptic connections.At 8 weeks after modeling,the Biotinylated dextran amine(BDA)tracing BDA tracing indicated that compared with the SCI group and the 3D-CC+BDNF group,the BDA positive CST fibers in the 3D-CC-BDNF group in the cross section(from rostral(-1 mm)to caudal(+1 mm))were significantly increased.At 8 weeks after modeling,TEM images showed that implantation of 3D-CC-BDNF could promote nerve fiber regeneration,axonal ingrowth,and myelinization of newly regenerated axons at the injury site.Conclusion:3D-CC-BDNF fabricated by low temperature extrusion 3D printing can promote nerve regeneration and functional recovery after the spinal cord is completely transected.Transplanting 3D-CC-BDNF at the injury site might be a potential treatment for clinical treatment of SCI.