The Construction Of Tissue Engineering Bone Based On Bio-electrospinning Technology

Part ? The preparation of P3HB4HB/(GE+PVA)composite scaffolds by coaxial electrospinning and its biocompatibilityObjective: This study seeks to prepare coaxial electrospun scaffolds of P3HB4HB/(Gelatin+PVA)with various concentration ratios and to identify scaffolds with advantageous properties.Methods: With 6% w/v P3HB4 HB as the core solution and an 8% w/v Gelatin(GE)+PVA mixture as the shell solution,the mass ratios(ing)of GE and PVA in each 10 m L shell mixture are 0.6:0.2(Group A),0.4:0.4(Group B)and 0.2:0.6(Group C).Coaxial electrospun scaffolds are prepared for using these three types of mixtures.The characterization,morphology,pore size,porosity,contact angle and tensile mechanical properties of these three scaffold groups are determined.Fourth-generation h ASCs are implanted into the scaffolds,and cell proliferation is measured by using the CCK8 assay.After the osteogenic and chondrogenic cells are induced and cultured for 14 days,the differentiation potential of the stem cells on the scaffolds is determined by alizarin red S,alkaline phosphatase(ALP),safranine O and alcian blue staining.Results: The pore size and porosity of the Group C scaffolds are better than those of Groups A and B(P<0.01).The ascending order of the tensile strength and modulus of elasticity is Group A<Group B<Group C,whereas the descending order of the nominal strain fracture is Group A>Group B>Group C.The CCK8 assay indicates that the cell proliferation rate of the Group C scaffolds is significantly higher than those of Groups A and B(P<0.01)at the ninth day.The scanning electron microscopy reveals that the pores in the scaffold derives from Group C that is consistent in size and interconnected,and the cells grow well in the scaffold.In contrast,the pores in the scaffolds derives from Groups A and B showing larger and uneven sizes.Moreover,the fiber diameters varies with fibrous fusion,and the interconnection among the pores is poor.Additionally,the number of cells growing in Group A is low.The number of cells growing in the Group C scaffold is significantly higher than those of Groups A and B.DAPI staining indicates that the cells adhere to the scaffold of each group,with the number of cells following the order Group C>Group B>Group A.The osteogenic and chondrogenic-specific staining shows that Group C is stronger than Groups A and B.Conclusion: When the mass ratio of PVA is 2:6,a P3HB4HB/(GE+PVA)composites scaffold with a core-shell structure can be prepared.The prepared scaffold has good biocompatibility and mechanical strength,suggesting that it may be an ideal scaffold for tissue engineering.Part ?The capacity of osteogenesis of human adipose-derived stem cells combined with P3HB4HB/GE+PVA biomimetic nanofibers in vivoObjective: The study is to investigate the potential of P3HB4HB/(GE+PVA)core-shell biomimetic nanofibers to produce neobone combined with differentiated human adipose-derived stem cells(h ASCs).Methods:the P3HB4HB/(GE+PVA)biomimetic three-dimensional scaffold is produced by coaxial electrospinning technique.The 4th generation of h ASCs is collected and inoculated into P3HB4HB/(GE+PVA)with concentration of 2 ×107/ml per scaffold,followed by the osteogenic differentiation medium.Noninductive groups are set as the contorl.The complex of cells and scoffolds is assessed by using SEM,TEM,fluorescence staining and DAPI staining.The induced and noninduced complexes of cells and P3HB4HB/(GE+PVA)scaffolds are implanted into the subcutaneous in the back of nude mics.The complexes of cells and scoffolds are terminated in vivo,assessed by HE staining,alizarin redstaining,von kossa staining,masson staining,collagentype I staining for 8 or 16 weeks after they are impanted into the vivo.Results: SEM shows that the nanofibrous is continuous as well as uniform,and the cell mass is uniform distribution of the nanofibers in each layer.The nanofibers clearly have core and shell structures,shown in the TEM image.No cytotoxicity is observed by the fluorescence staining and DAP Istaining.After 14 days in vitro culture,the differentiated h ASCs-P3HB4HB/(GE+PVA)nanofibers scaffold is implanted into the subcutaneous layer of nude mice for 8 or 16 weeks,non-differentiatedh ASCs-P3HB4HB/(GE+PVA)nanofibers scaffold is implanted as the control group.The implant of differentiated h ASCs-P3HB4HB/(GE+PVA)forms bone-like tissue after 16 weeks of implantation,and stains positive for alizarin redstaining,von kossa staining,masson staining,collagentype Istaining.Conclusion:human adipose-derived stem cells combined with P3HB4HB/(GE+PVA)scaffolds induced in vitro have the capability of heterotopic osteogenesis in vivo.Part ? The capacity of osteogenesis of P3HB4HB/(GE+PVA)-h ASCs in vivoObjective: The goal of this study is to investigate the potential of P3HB4HB/(GE+PVA)core-shell nanofibers and produce neobone upon coaxial bioelectrospinning simultaneously with differentiated human adipose-derived stem cells(h ASCs).Methods: The nanofibers scaffolds are prepared by coaxial bio-electrospinning P3HB4HB/(GE+PVA)-h ASCs in vitro with or without osteogenic media for 14 days.The complex of P3HB4HB/(GE+PVA)-h ASCs is assessed by using SEM,fluorescencestaining and DAPI staining.The induced and noninduced complexes of P3HB4HB/(GE+PVA)-h ASCs are implanted into the subcutaneous in the back of nude mics.The complexes of P3HB4HB/(GE+PVA)-h ASCs are terminated in vivo,assessed by using HE staining,alizarin red staining,von kossa staining,masson staining and collagentype Istaining at 8 or 16 weeks after they are impanted into the vivo.Western-blot is used to detect the expression of collagen type? and osteocalcin.Results: Scanning electron microscopy shows that the nanofibrous is continuous as well as uniform,and the cell mass is uniform distribution of the nanofibers in each layer.The differentiated cells produce abundant extracellular matrices with increasing culture time.No cytotoxicity is observed by the fluorescence staining and DAPI staining.After 14 days in vitro culture,the differentiated P3HB4HB/(GE+PVA)-h ADSCsnanofibers scaffold is implanted into the subcutaneous layer of the nude mice for 8 or 16 weeks,non-differentiated P3HB4HB/(GE+PVA)-h ASCs nanofibers scaffold is implanted as the control group.The differentiated P3HB4HB/(GE+PVA)-h ASCs implants form bone-like tissue after16 weeks of implantation,and stain positive for alizarin red staining,von kossa staining,masson staining and collagentype Istaining.The experimental group of western blot is used to detect the expression of type ? collagen and low expression of osteocalcin in nude mice within 8 weeks.The strong expression of type? collagen and osteocalcin is expressed for 16 weeks.The control group,the expression of type? collagen and osteocalcin are not expressed in nude mice within 16 weeks.Conclusion: This study demonstrates that coaxial bio-electrospinning of cells and materials can not only produce the scaffolds complication and nanocrystallization,but also realize the precise cultivation of cells,which is a suitable material for the tissue engineering of bone.