Composite Construction Of Tissue Engineering Bone By Multilayer Electrospinning Of HBMSCs And Polyvinyl Alcohol/Poly (3-Hydroxybutyrate-Co-4-Hydroxybutyrate)

Part ?The preparation of P3HB4HB/PVA composite scaffolds by Multilayer electrospinning and its biocompatibilityObjective: Objective: to prepare PVA/P3HB4 HB Multilayer electrospinning scaffolds,and to investigate the physical properties and biocompatibility of scaffolds in vitro.Methods: Put the human anterior superior iliac spine marrow to isolation and culture of human bone marrow mesenchymal stem cells.Prepare 6% w/v P3HB4 HB and PVA solution respectively.prepare the coaxial electrospinning scaffold with P3HB4 HB solution?PVA solution and PVA/P3HB4 HB solution respectively(marked as A ? B and C).Detect the morphology and characterization,contact angle,and the tensile mechanical properties of the scaffold.The fourth generation of BMSCs were seeded on scaffolds,detected cell adhesion rate ?cell proliferation and acridine orange fluorescent staining;cells combined with scaffolds,cultured the osteoblasts and chondrocytes for 14 d,using alizarin red and toluidine blue staining to verify BMSCs' differentiation potential.Results:Scanning electron microscopic observation of the three groups of scaffold,shows the structure is three-dimensional network and connects with each other.between A and C group,the fiber diameter is homogeneous and ordered;the tensile strength,tensile modulus and maximum stress of the fiber scaffold were: the A group < the B group<C group(p<0.05).Compositely Cultured The cell with the scaffold,cell adhesion rate of the 1,3,6h is B group >C group >A group(P < 0.05);CCK8 assay showed that the proliferation of cells in A group were significantly lower than those of B,C group at 1,3,5 and 7d.When cultured for 1-4 days,the cell proliferation of B group is greater than the C group,after 4 days,the cell proliferation rate of group C was faster than that of group B(P < 0.05);Compositely Cultured The cell with the scaffold for 7 days,stained through acridine orange fluorescent,could see green fluorescent cells in the scaffold.cells of B and C groups were more than A group.After induction culture,the specific staining of bone formation and cartilage formation of C group was stronger than that of A and B group.Conclusion: the electrospun PVA/P3HB4 HB scaffolds have good biocompatibility and mechanical strength,which is expected to be an ideal tissue engineering scaffold.Part ? composite construction of tissue engineering bone by multilayer electrospinning of h BMSCs and polyvinyl alcohol/poly(3-hydroxybutyrate-co-4-hydroxybutyrate)Objective: prepare hBMSCs-PVA/P3HB4 HB coaxial electrospinning scaffolds composite,and to investigate the osteogenic ability of the composite PVA/P3HB4 HBelectrical scaffold of h BMSCs in nude mouse.Method: Extracted and cultured primary h BMSCs from the bone marrow.The surface antigens expression of the 4th passage cells were detected by flow cytometry.the fourth passage cells suspension was inoculated to the chip,cultured the osteoblasts for 14 days using alizarin red staining,alkaline phosphatase staining and type I collagen immunohistochemical staining to detect the osteogenetic differentiation.Prepare 6%w/v P3HB4 HB and PVA solution respectively,the experimental group prepare h BMSCs-PVA/P3HB4 HB coaxial electrospinning scaffolds composite,the control group drip the cells on scaffolds.Culture cell-scaffolds in vitro for 1,7 days and observe the growth of cells on the scaffold by scanning electron microscopy(sem)and DAPI fluorescence staining.Both groups osteogenesis induced in vitro for 14 days,subcutaneously implanted into nude mice,remove the implant in 1,3,6 months after implanted and do osteogenesis related dyeing.Results:Flow cytometry indicated that the 4th passage cells not expression of CD34,CD45,HLA-DR,but high expression of CD44,CD73,CD90 and CD105.after osteogenesis induction,alizarin red staining,alkaline phosphatase staining,collagen type ? immune staining were all positive.Scanning electron microscopic observation of the three groups of scaffold,shows the structure is three-dimensional network and connects with each other,the fiber diameter is homogeneous and ordered.cells adhered to the fiber and grew,secretory cell matrix,the microscopic observation showed the number of cells in 7d was more than 1d,the experimental group was more than the control group.DAPI dying showed that the cells planting on the scaffold uniformly on the scaffold.subcutaneously implanted into nude mice,remove the implant in 1,3,6months after implanted and all of the alizarin red staining,Von Kossa staining,mason trichromatic dyeing,collagen type ? immunohistochemical staining test were positive,the control group was weakly positive.Conclusion: multi-layer electrospinning PVA/P3HB4 HB scaffold is suitable for cell growth,has the development potential to use as bone tissue engineering scaffold;A molding multilayer electrospinning h BMSCs-PVA/P3HB4 HB complex,after osteogenesis induced in vitro,it has the ability of constructing tissue engineering bone ectopicly in nude mice.