| Objective:The development of functional materials to improve or restore the biological functions of damaged tissues plays an extremely important role in the fields of regenerative medicine and tissue engineering.Research:(1)Coaxial electrospinning technology to construct a transitional structured(Aligned-Lattived)bionic fiber support,and to evaluate its physical and chemical properties.(2)Explore the ability to construct tissue engineered bone in a rat skull defect model of MC3T3-E1 cell composite Lattived fiber scaffold.(3)To explore the ability of Aligned fiber scaffolds to promote adhesion,proliferation and tendon differentiation of bone marrow mesenchymal stem cells(BMSCs),as well as to repair defects in rat tendon-bone interface model after in vitro induction of composite Aligned-Lattived bionic fiber scaffolds.Methods:(1)Using coaxial electrospinning technology,aligned fiber scaffolds were constructed with poly(3-hydroxybutyrate-co-4-hydroxybutyrate)(P34HB)as core solution and type I collagen(Col I)as shell solution;Lattived fiber scaffolds were constructed with P34HB-polyethylene glycol(PEG)as the shell solution and polyvinyl alcohol(PVA)-bone morphogenetic protein-2(BMP-2)polypeptide as the core solution,micron fibers with hollow porous structure were constructed.The morphology of Aligned-Lattived fiber scaffolds were characterized by scanning electron microscope(SEM),Fourier transform infrared spectroscopy(FIRT)was used to analyze the changes of chemical groups and chemical bonds of the scaffold,water contact angle instrument was used to analyze the hydrophilic properties of the scaffold surface,test of mechanical properties of Aligned-Lattived fiber scaffolds,and ELISA was used to detect the cumulative release of BMP-2 within 28 days.(2)Lattived fiber scaffolds were constructed by coaxial electrospinning technology,and MC3T3-E1 cells were cultured at 2.5×10~4/well was inoculated on the surface of the material in the 24 well plate,which was divided into PVA/P34HB-PEG group and PVA-BMP-2/P34HB-PEG group.DAPI staining,CCK-8,Live/dead staining and SEM were used to verify the biocompatibility of fiber scaffolds;The osteogenic differentiation ability of fiber scaffolds was identified by ARS staining and quantitative analysis,ALP quantitative analysis and OCN immunofluorescence staining;MC3T3-E1-Lattived fiber scaffold complex was implanted into the rat skull defect model in the induction group and non-induction group.The specimens were taken for Micro CT and histological staining(HE staining and Masson staining)at 4and 8 weeks respectively.(3)Aligned fiber scaffolds were constructed by coaxial electrospinning technology,and BMSCs were cultured at 2.5×10~4/well was inoculated on the surface of the material in the 24 well plate and divided into P34HB group and P34HB/Col I group.DAPI staining,CCK-8,Live/dead staining and SEM were used to verify the biocompatibility of fiber scaffolds;q RT-PCR was used to identify the tendon differentiation ability of aligned fiber scaffolds;BMSCs in the induced group and non-induced group were implanted into the Achilles tendon insertion defect model of rats.The specimens were taken at 6 and 12 weeks for HE staining,Masson staining and Sirius staining,as well as TEM observation.Results:(1)SEM observation shows the aligned and latticed arrangement of Aligned-Lattice fiber scaffolds respectively.Lattice fiber scaffolds show hollow porous structure,and TEM shows the core-shell structure of fibers.FIRT results showed that the characteristic peak of Col I appeared in the P34HB/Col I fiber scaffold after coaxial electrospinning,and the characteristic peak of Col I disappeared within 30 min before cross-linking,while the characteristic peak of Col I remained unchanged after cross-linking.The PVA/P34HB-PEG fiber scaffold groups remained stable after coaxial electrospinning.The contact angle of P34HB/Col I composite fiber membrane was smaller than that of simple P34HB fiber membrane,and the hydrophilicity was better,the difference was statistically significant(P<0.05).The mechanical test results showed that the tensile strength and maximum tensile strength of the Aligned fiber scaffold were significantly better than those of the Lattived fiber scaffold.However,the Young’s modulus of Lattived fiber scaffold was higher than that of Aligned fiber scaffold,and the difference was statistically significant(P<0.05).ELISA results showed that BMP-2 was released continuously and slowly on PVA-BMP-2/P34HB-PEG scaffolds.(2)DAPI staining showed that MC3T3-E1 cells could adhere to the two groups of fiber scaffolds,and CCK-8 showed that the cells could proliferate and grow on the two groups of scaffolds,especially on PVA-BMP-2/P34HB-PEG fiber scaffolds.The difference between the two groups was statistically significant(P<0.05);Live/dead staining showed that most cells survived on the scaffolds of the two groups,with only a small number of dead cells;SEM results showed that the cells could adhere and spread on the two groups of scaffolds,and the cells on PVA-BMP-2/P34HB-PEG fiber scaffolds secreted more extracellular matrix;The results of ARS staining and quantitative analysis,ALP quantitative analysis and OCN immunostaining showed that the fiber scaffold containing BMP-2 polypeptide had better osteogenic differentiation potential,and the difference between the two groups was statistically significant(P<0.05);Micro CT results showed that in PVA-BMP-2/P34HB-PEG group,new bone tissue was formed in the defect area at 4 weeks.With the extension of time,most of the bone defect area was covered by new bone at 8 weeks.BV/TV and BV values were better than those in PVA/P34HB-PEG group.There was significant difference between the two groups(P<0.05);HE staining showed that regenerated bone with typical mature bone structure was formed in the central part of fiber scaffold in PVA-BMP-2/P34HB-PEG group,and some new bone grew at the edge of bone defect in PVA/P34HB-PEG group.After 8 weeks of implantation,the repair area of PVA-BMP-2/P34HB-PEG group found large plate bone tissue formation and small vessel proliferation,which was better than that of the control group.The remaining material scaffolds were detected in both groups.It was observed that the collagen fiber content in the experimental group was better than that in the control group at 4 weeks,and the collagen fiber content in the experimental group was better than that in the control group at the same time.(3)DAPI staining showed that BMSCs could adhere to the two groups of fiber scaffolds.CCK-8 results showed that cells could proliferate and grow on the two groups of scaffolds,especially on P34HB/Col I fiber scaffolds.There was significant difference between the two groups(P<0.05);Live/dead staining showed that most cells survived on the scaffolds of the two groups,with only a small number of dead cells;SEM results showed that the cells could adhere and spread on the two groups of scaffolds,and the cells on P34HB/Col I fiber scaffolds secreted more extracellular matrix;The results of q RT-PCR showed that the fiber scaffold modified by Col I had better tendinous differentiation potential,and the difference between the two groups was statistically significant(P<0.05);Histological staining showed that new bone tissue and a small amount of collagen fibers appeared in the interface area of the experimental group at 6 weeks.After 12 weeks of implantation,the regenerated tissues in both groups became more mature.Compared with the control group,non/mineralization cartilage formation and calcification and better arrangement of collagen fibers were observed in the interface area of the experimental group.TEM results showed that the ultrastructure of collagen fibers in the experimental group was better than that in the control group,which was similar to the hierarchical structure of natural tendon.Conclusions:(1)Coaxial electrospinning technology construction transitional structured(Aligned-Lattived)fiber scaffold has the bionic structure of tendon-bone interface,good hydrophilic,certain mechanical strength,slow release of BMP-2 polypeptide.(2)PVA-BMP-2/P34HB-PEG fiber scaffold has good biocompatibility,supports MC3T3-E1 cell adhesion,diffusion and proliferation,and can induce MC3T3-E1 cell differentiation toward osteogenesis,proving that PVA-BMP-2/P34HB-PEG is a potential new guide bone tissue regeneration and repair material.(3)Coaxial electrospinning technology to construct Aligned-Lattived bionic fiber scaffold,which was confirmed by in vitro cell experiments and in vivo animal experiments,and is expected to become a potential new tissue engineering material for guide tendon-bone interface repair. |
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