Biomimetic Biodegradable Scaffold Loaded Human Wharton's Jelly-derived Mesenchymal Stem Cells To Construct Tissue Engineering Annulus Fibrosus

Objective: 1.To evaluate the basic characteristics,degradation rate and mechanical properties of degradable tissue engineering annulus fibrosus(AF)scaffolds that was prepared by melt spinning using polycaprolactone(PCL),poly(lactic-co-glycolic acid)(PLGA)and a mixture of the two.2.To study the degradation performance of biomimetic degradable scaffolds and the inflammatory response to surrounding tissues.3.To explore the feasibility of biodegradable scaffold loaded Human Wharton's jellyderived mesenchymal stem cells(HWJ-MSCs)to construct biomimetic tissue engineering annulus.Methods: 1.Bionic fiber scaffolds were prepared by melt spinning technique using PCL,PLGA and a mixture of different ratios(PLGA: PCL 20/80,35/65,50/50,65/35,80/20).The PCL scaffold served as a control group.The PLGA in the scaffold obtained was dissolved using dimethylformamide(DMF)to verify the scaffold material composition ratio and material mixing degree.The structure of the scaffold was observed by a stereo microscope and a scanning electron microscope(SEM),and the fiber diameter,porosity,and pore sizer of the scaffold were measured.The compressive and tensile elastic moduli were measured using a mechanical loading device.2.The in vivo microenvironment was simulated in vitro,and the degradation process of each group of scaffolds was observed and the degradation rate in vitro was measured.The scaffolds were implanted into the skin of rats,and the degradation rate of the scaffolds in each group was observed and detected.The levels of inflammatory factors in the tissues surrounding the scaffolds were detected by ELISA kit.3.The primary HWJ-MSCs were isolated and cultured by human umbilical Wharton gel.After passage,the P3 cells were inoculated into each group of fiber scaffolds for cultivation.Live-dead staining,phalloidin cytoskeleton staining and CCK-8 analysis of cell adhesion,morphology and proliferation on the scaffold.The biomimetic scaffold-HWJ-MSCs complex was induced to culture in a fiber-like ring induction solution for 6 weeks,and the expression of type I,II collagen and Aggrecan protein was detected by Western-blot.Results:1.The scaffold has a white hollow ring-shaped with an inner diameter of 2 mm and an outer diameter of 6 mm.The outer circumference can be seen as regular rhombic pores surrounded by 60° angle fibers,and the cross-section filaments are evenly distributed around.By dissolving the obtained PLGA treatment,the material ratio of the obtained scaffold was the same as that of the raw material,and there was no significant change in the scaffold structure before and after the treatment.The basic characteristics of the pure PCL scaffold are fiber diameter(49.67±1.35)?m,pore size(71.52±1.13)?m and porosity(73.14±0.77)%,compressive elastic modulus(2.36±0.19)MPa,and tensile modulus of elasticity(8.95±0.22)Mpa;the scaffold fiber diameter of the mixed material group(PLGA:PCL-80/20?65/35?50/50?35/65?20/80)was(50.42±1.69?50.95±1.48?50.48±1.62?50.91±1.37?50.31±1.20)?m,the pore size was(70.37±1.109?71.51±1.83?70.51±1.07?70.29±1.16?70.47±1.01)?m,the porosity was(72.64±0.73?72.91±0.45?73.56±0.73?72.34±0.81?73.41±1.19)%,and the compressive elastic modulus was(0.03±0.01?0.09±0.01?0.32±0.07?1.42±0.11?2.07±0.12)MPa,and the tensile elastic modulus is(0.05±0.02?0.14±0.05?3.04±0.19?5.47±0.23?7.06±0.25)Mpa,respectively.2.The weight loss of PCL group was not statistically significant after three months of in vitro degradation and the mass residual rates in each mixed material group(PLGA:PCL-50/50?35/65?20/80)was(61.63±0.91?78.56±0.89?85.01±0.84)%.After three months of in vivo degradation,the mass residual rates in each mixed material group(PLGA:PCL-50/50?35/65?20/80)were(34.01±1.74,56.29±1.52,85.01±0.84)%.The degradation rate of the scaffolds in each mixed material group was significantly faster than that in vitro,and the structure remained intact.No fragmentation was observed.3.The 7-day Live-dead staining and CCK-8 assays of biomimetic scaffold composite HWJ-MSCs showed that the higher the PLGA gravity in the hybrid scaffold,the higher the viable cell fluorescence and the higher the OD value(450 nm).The cytoskeletal staining of the phalloidin showed that the cells of each group showed a long fusiform extension.Western-blot assay showed that the amount of type I collagen and Aggrecan protein secreted by cells in the mixed material scaffolds at each time point was higher than that in the PCL group(P<0.05),and the content of type I collagen and Aggrecan protein in each group increased with time.obviously increase.Conclusion:1.The biomimetic PCL-PLGA degradable annulus fibrosus scaffold was constructed by melt spinning method.The fiber orientation was good,the fiber diameter was uniform,and the porosity and pore size of the scaffold were suitable.The compression modulus and tensile modulus of the three groups of PLGA-50,PLGA-35 and PLGA-20 are close to the mechanical range of the human annulus fibrosus,and have the potential as an ideal annulus fibrosus tissue engineering scaffold.2.Mixing PLGA into PCL at different ratios can effectively increase the degradation rate and reduce the tissue inflammation around the scaffold implantation site.After the first stage of experimentation,the rate of degradation of the two groups of PLGA-35 and PLGA-20 in the remaining mixed material scaffold was compatible with the known rate of repair of the annulus.3.Mixed material scaffolds prepared by mixing PLGA with PCL in different proportions can enhance the proliferation capacity,cell activity and extracellular matrix secretion of hwj-mscs on the scaffolds.After the second stage experiment was used to exclude the PLGA-50 scaffold,the PLGA-35 scaffolds in the remaining mixed material scaffolds were more advantageous in improving cell proliferation,cell viability and extracellular matrix secretion after compound seed cells.