| Objective:1.To investigate the feasibility of fabricating 60 ° angle-ply multi-lamellar scaffold by melt-spinning for annulus fibrosus(AF)tissue engineering.2.To explore the feasibility of constructing tissue engineering annulus fibrosus by 60 ° angle-ply multi-lamellar scaffold with Human Wharton's jelly-derived mesenchymal stem cells(HWJ-MSCs)in vitro.3.To investigate the effect of fiber diameter on the induced differentiation of Human Wharton's jelly-derived mesenchymal stem cells.Methods:1.A new annulus fibrosus scaffold was fabricated by innovative melt spinning strategy using poly(?-caprolactone)particles.Stereomicroscope and SEM was used to observe the surface morphology and fiber diameter and pore size of scaffold.Micro-CT was used to analyze the porosity of scaffold.Mechanical testing system was used to analyze the compressive elastic modulus(Young's modulus)of the scaffold.2.HWJ-MSCs were isolated from umbilical and cultured to P3 generation were seeded on the scaffold,then induction culture for a period of time.The scaffold was evaluated by Stereomicroscope,SEM and Micro-CT.The cell viability,attachment,proliferation and infiltration were evaluated by SEM,live/dead,fitc-phalloid staining.The expression of proteoglycan,collagen type I and collagen II gene were detected by RT-PCR and the expressions of collagen type I,collagen II protein and aggrecan protein were detected by Western-blot.3.Preparation of three different diameter scaffold,inoculated Human Wharton's jelly-derived mesenchymal stem cells.The stereospectrophotometer and SEM were used to observe the scaffold structure and the cell adhesion and secretion matrix.The compressive and tensile modulus of the three scaffolds was measured.The gene and protein expression of the scaffolds cultured for 7 days and 21 days were detected by RT-PCR and Western-blot.Results:1.Scaffold was a white ring,inside diameter was 2mm and outside diameter was 7mm,between the side of the wire angle of 60°was a regular diamond,fiber evenly distributed around.The diameter of the scaffold was(42.95 ± 1.88)?m,the pore diameter was(74.94 ± 3.73)?m,the porosity was(58.25 ± 0.11)%,the compressive elastic modulus was(1.73 ± 0.44)MPa and the tensile modulus was(7.75 ± 0.38)MPa.2.Stereomicroscope to see the shape of the natural fiber ring complex;SEM see the cell spherical or short spindle adhesion to the scaffold;live and dead staining showed good cell activity,no dead cells;FITC staining showed cytoskeleton arranged along the fiber direction;Three weeks later,the staining results were better in the induction group than in the control group;The results of Western-blot showed that the cell protein was significantly increased and the secretion of extracellular matrix was gradually increased(P<0.05).RT-PCR The results showed that the expression of collagen type?and collagen type?and proteoglycan gene increased gradually(P<0.05).3.The diameters of the fiber in the microscope were different.SEM showed that a large amount of cell matrix was secreted by the three scaffolds.Quantitative analysis showed that the gene and protein increased correspondingly,and the gene with a fiber diameter of(42.95±1.88)?m expressed better protein,Compression and tensile modulus of elasticity increase with increasing incubation time.Conclusion:1.The use of melt spinning method to construct a fiber ring stent,pore size,good porosity and moderate mechanical properties close to the natural fiber ring,tissue engineering fiber ring is an ideal carrier.2.After in vitro induction of fused human wharton's jelly-derived mesenchymal stem cells with 60°angle-ply multi-lamellar annulus fibrosus scaffold,the composite tissue with similar structure,biochemical composition,mechanical properties and human natural annulus fibrosis can be obtained.3.By qualitative and quantitative analysis of the fiber diameter(42.95±1.88)?m stent,human umbilical cord cells induced differentiation is better,the resulting composite tissue is suitable for repair of annulus fibrosus defects. |
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