Study On Construction Of Tissue Engineering Annulus Fibrosus With Bionic Biodegradable Scaffold

Objective: 1.Evaluate the feasibility of polycaprolactone(PCL)and polydioxanone(PDS)by melt spinning to fabricate bionic biodegradable intervertebral disc(IVD)annulus fibrosus scaffold.2.To study the feasibility of fabricate bionic biodegradable tissue engineering scaffolds compound with Human Wharton's Jelly Mesenchymal Stem Cells(HWJ-MSCs).Methods: 1.Five groups of bionic scaffolds with different proportions were fabricated by melt spinning using PCL,PDS and their mixtures as raw materials,including PCL group,PCL/PDS70/30 group,PCL/PDS50/50 group,PCL/PDS30/70 group and PDS group.The macroscopic and microscopic structure of the scaffolds were observed by Stereomicroscope and scanning electron microscopy(SEM).The fiber diameter,pore size and porosity of the scaffolds were measured.Mechanical measuring instrument was used to analyze the mechanical properties.The hydrophilicity was measured by contact angle measuring instrument.Observing the biodegradable of the scaffold in vitro and in vivo.The expression of inflammatory factors around the tissue was detected by enzyme-linked immunosorbent assay(ELASA).2.HWJ-MSCs cells were isolated and cultured,seeded on the scaffold.CCK-8 assay,SEM,live/dead staining,phalloidin staining were evaluated the cell viability,attachment,proliferation and infiltration..After 1 week and 3 weeks of induction culture,the expression of type I,II collagen and proteoglycan proteins was detected by Western blot.Result: 1.The scaffold was hollow circular,milky white which outer diameter 6mm and inner diameter 2mm.Stereomicroscope and SEM results showed that the scaffold were banded and ring parallel orientation,and has smooth surface.The fiber diameter was 45-55?m,pore size was 70-75?m.The mechanical properties test results show that the pure PDS group are the worst,the PCL group are the best.The mechanical properties of the other three scaffolds groups gradually become stronger as the proportion of PCL increases.The PDS group is the most hydrophilic,and the PCL group is the least hydrophilic.The hydrophilicity of the three PCL/PDS groups gradually decreased with the increase of PCL ratio.Biodegradable test results showthat the quality of the five groups scaffold is related with time closely.At the 12 th week,the biodegradable rates in vitro and in vivo were 21.44% and 32.89% in PCL/PDS70/30 group,32.29% and 38.08% in PCL/PDS50/50 group,36.81% and46.90% in PCL/PDS30/70 group,40.70% and 61.11% in PDS group,respectively.The PCL group has almost no biodegradable in vitro and in vivo.The inflammatory response caused by the PDS group and the PCL/PDS hybrid groups were lower than that of the PCL group.The higher the PCL ratio,the more severe the inflammatory response.2.The P3 HWJ-MSCs cultured in vitro were long fusiform shaped.After the cells were compound with the scaffold for 7 days,CCK-8 assay showed good cells attactment and proliferated.The best cell proliferation in PCL/PDS50/50 group.Live/dead staining showed that the scaffold was non-cytotoxic to cells.SEM showed that the cell adhesion on the scaffold,and secretion a large amount of extracellular matrix.Phalloidin staining observation showed that the cells adhered to the scaffold in a long fusiform shape.Western blotting showed that type I collagen and proteoglycan by each group of scaffold cells increased significantly with time over.The amount of type I collagen and proteoglycan in PCL / PDS 70/30 and PCL / PDS50/50 were higher than PCL after 1 week and 3 weeks(P <0.05).Conclusion: 1.Using PCL and PDS by melt spinning to fabricate bionic biodegradable IVD annulus fibrosus scaffold have suitable biodegradable rate,superior mechanical properties and good biocompatibility,which make it a suitable candidate for tissue enineering annulus fibrosus scaffold.2.PCL/PDS biodegradable scaffolds compound with HWJ-MSCs has good biological activity and can be used to construct biomimetic biodegradable tissue engineering annulus fibrosus scaffold.