Study On Silk Fibroin Integrated Scaffolds With Biomimetic Microchannels For Regeneration Of Intervertebral Disc Degeneration

Objective: 1.Silk fibroin integrated scaffolds with angle-ply multi-lamellar microchannel annulus fibrosus(AF)and porous nucleus pulposus(NP)were fabricated by reverse poreforming method;2.To detect the biocompatibility of the biomimetic microchannel silk fibroin integrated intervertebral disc scaffold and observe the regulation effect of its microchannel structure on cell behavior in vitro;3.Subcutaneous implantation was used to evaluate the biocompatibility of the biomimetic microchannel silk fibroin integrated intervertebral disc scaffold and observe the guiding effect of its microchannel structure on tissue infiltration;4.To evaluate the tissue integration and repair ability of the biomimetic microchannel silk fibroin integrated intervertebral disc scaffold by total disc replacement of rat tail.Method: 1.PCL was used to fabricate the ±30° angle-ply multi-lamellar sacrificial mold for AF by melt spinning technology,and the paraffin microspheres were used as pore-forming mold for NP,and then infuse 15% silk fibroin solution into the space of the mold.PCL and paraffin were leached out with chloroform and n-hexane after freeze-drying.The silk fibroin integrated intervertebral disc scaffold with angle-ply multi-lamellar microchannel AF and porous NP was successfully prepared.Scanning electron microscope(SEM)and micro-CT were used to observe the microstructure of the scaffold and measure the inner diameter of the microchannel in the AF area and pore size in the NP area.The porosity of the scaffold was determined by mass volume method.The chemical composition of the scaffold was analyzed by Raman spectroscopy,and mechanical testing system was used to determine the mechanical properties.2.3T3 cells were seeded onto the surface of the sterilized scaffold,and continuously cultured for some days in a normal cell incubator.Live/dead staining was used to evaluate the cytocompatibility of the scaffold and observe the migration of cells inside the scaffold.CCK-8 was used to detect the proliferation of cells on the scaffold. Phalloidin staining and SEM were used to characterize the cell morphology in both AF and NP area.3.Sterilized scaffolds were implanted under the skin of rats,and were taken out at 2,4 and 8weeks after operation,fixed with 4% paraformaldehyde,dehydrated with gradient ethanol,paraffin-embedded and were cut into sections with 4?m thick.HE staining was used to observe the degree of tissue infiltration and vascularization inside the scaffold;Immunofluorescence staining of macrophage-specific markers(CD68,CD206,i NOS)was used to evaluate the biocompatibility of the scaffold in vivo.4.The biomimetic intervertebral disc scaffold and the control scaffold were implanted into the caudal spine of rats,and then the rats were sacrificed at 2,4 and 12 weeks after operation.The caudal spine samples were stored in two ways: Some samples were frozen in-80? for mechanical testing,and other samples were fixed with 4% paraformaldehyde and then decalcified with EDTA decalcification solution,and were cut into sections with 4?m thick after conventional paraffin embedding.HE staining was used to observe the integrate growth of scaffolds with the native vertebrae;Sirius red staining and Alcian blue staining were used to analyzed the chemical composition of neo-tissues in the rat caudal spine disc.Result: 1.SEM and micro-CT images showed that the biomimetic microchannel silk fibroin integrated intervertebral disc scaffold was characterized with biomimetic microchannel structure,which composed of angle-ply multi-lamellar microchannel AF and porous NP.The microchannel inner diameter of the AF was 110±15?m,and the pore size of the NP was 170±22?m.The porosity of the biomimetic scaffold was 89.33±6.58%,and the porosity of the control scaffold was 69.52±5.25%.The Raman spectroscopy showed that the material composition of the biomimetic scaffold was pure SF material;Mechanical properties testing showed that the compression modulus of the biomimetic scaffold was 3.12±0.25 Mpa.2.Live/dead staining indicated that 3T3 cells can easily adhere to the scaffold surface and survive well;FITC-Phalloidin staining and SEM results showed that the cells exhibited different morphologies in different microchannel structure of AF and NP.3.HE staining of the scaffold after subcutaneous implantation showed that the biomimetic intervertebral disc scaffold and the control scaffold could integrate well with the subcutaneous tissue.The subcutaneous tissue could grow into the biomimetic intervertebral disc scaffold through microchannels,and many blood vessels can be seen,while subcutaneous tissue was difficult to grow into the control scaffold;Immunofluorescence staining of macrophage-specific markers showed that there were many M1 macrophages and a small amount of M2 macrophages in the biomimetic scaffold at 2 weeks.In comparison,the number of M1 macrophages decreased significantly while the number of M1 macrophages increased significantly at 4 weeks.4.Biomimetic intervertebral disc scaffolds and control scaffolds can partially maintain intervertebral body height after implantation into rat caudal spine.The MRI images of rat tail demonstrated that the implanted biomimetic intervertebral disc scaffold and control scaffold showed bright signal after operation,while the bright signal disappeared in the segment of discectomy.In addition,mechanical tests of the rat tail motion segments at 4 and 12 weeks after operation showed that biomimetic intervertebral disc scaffold and control scaffold could both partially restore the biomechanical properties of the rat tail,and the biomimetic intervertebral disc scaffold implanted group showed better mechanical property than the control scaffold group.HE staining revealed that the biomimetic intervertebral disc scaffold and the control scaffold could fused with adjacent vertebral body tissues and partially restored intervertebral disc height after implantation in rat tail.Sirius red and Alcian blue staining showed that the components of neo-tissue between vertebral body were mainly Collagen I and a small amount of proteoglycan.Conclusion: 1.A novel biomimetic microchannel silk fibroin integrated intervertebral disc scaffold was successfully fabricated,which possessed similar spatial structure and mechanical properties to natural intervertebral disc.2.The biomimetic microchannel silk fibroin integrated intervertebral disc scaffold possessed good biocompatibility,and its different biomimetic microchannel structure in AF and NP could guide cells to exhibit different growth morphology.3.In situ repair experiment of rat tail intervertebral discs showed that the biomimetic intervertebral disc scaffold can fuse with the adjacent vertebral tissue and it can sustain the disc height and the mechanical force of the rat tail,which provide a potential tissue engineering method for the regeneration and repair of degenerated intervertebral disc.