The Research And Development Of Bio-Electrospinning Technology And Construction Of Tissue Engineered Bone

Part ? Isolation, culture and osteogenic differentiation of human bone marrow mesenchymal stem cells in vitroObjective: To isolate and culture human bone marrow mesenchymal stem cells and evaluate their potential of osteogenic differentiation in vitro. Methods: h BMSCs were isolated and purified by adherence screening culture. The characteristics and morphology of h BMSCs were observed inverted microscopically and histochemically. The CD44, CD90, CD105, CD73, CD34, CD45 and HLA-DR expression of the 4th passage cells were detected by flow cytometry. Then under certain inductive conditions, h BMSCs were made to differentiate into osteoblasts, and identified in terms of Alizarin red, alkaline phosphatase and collagentype I staining respectively. Results: The cells cultured with this technique posses a general bionomics of h BMSCs, appeared morphologically as spindle and colony-shape. Flow cytometry indicated that the 4th passage cells not expression of CD34, CD45 or HLA-DR, but high expression of CD44, CD90, CD105 and CD73. The extracellular matrix was stained positively for alizarin red, alkaline phosphatase and collagentype I after initial osteogenic induction. Conclusion: The method of isolation and culture of h BMSCs is simple and feasible, by which h BMSCs can be effectively isolated and purified. Furthermore, h BMSCs have proliferation and osteogenic differentiation ability.Part ? The capacity of osteogenesis of bone marrow mesenchymal stem cells combined with P3HB4HB/PVA biomimetic nanofibers in vivoObjective: To investigate the potential of P3HB4HB/PVA core-shell biomimetic nanofibers to produce neobone combined with differentiated human bone marrow mesenchymal stem cells(h BMSCs). Methods: Fabrication of P3HB4HB/ PVA biomimetic three-dimensional scaffold by coaxial electrospinning technique. The 4th passage h BMSCs were collected and inoculated into P3HB4HB/PVA with concentration of 2 ×107/ml per scaffold, followed by the osteogenic differentiation medium. Noninduction groups were set as the contorl. The complex of cells and scoffolds were assessed by using SEM, TEM, water contact angle, fluorescence staining and DAPI staining. The induces and noninduced complexes of cells and P3HB4HB/PVA scaffolds were implanted into the subcutaneous in the back of nude mics. The complexes of cells and scoffolds were terminated in vivo, assessed by using HE staining, alizarin red staining, von kossa staining, masson staining, collagentype I staining at 8, 16 weeks after they were impanted in vivo. Results: SEM showed that the nanofibrous were continuous and uniform, cell mass and uniform distribution of the nanofibers in each layer. The nanofibers clearly had core and shell structures, as shown in the TEM image. According to the membranes were tested for water contact angle, the P3HB4HB/PVA nanofibers was more hydrophilic than the fibers composed of solely P3HB4HB(P<0.05). No cytotoxicity was observed by the fluorescence staining and DAPI staining. After 14 days of in vitro culture, the differentiated h BMSCs-P3HB4HB/PVA nanofibers scaffold were implanted into the subcutaneous layer nude mice for 8 or 16 weeks, non-differentiated h BMSCs-P3HB4HB/PVA nanofibers scaffold were implanted as the control group. The differentiated h BMSCsP3HB4HB/PVA implants formed bone-like tissue after 16 weeks of implantation, and stained positive for alizarin red staining, von kossa staining, masson staining, collagentype I staining. Conclusion: Human bone marrow mesenchymal stem cells combined with P3HB4HB/PVA scaffolds induced in vitro have the capability of heterotopic osteogenesis in vivo.Part ? The capacity of osteogenesis of P3HB4HB/PVA-h BMSCs in vivoObjective: The goal of this study was to investigate the potential of P3HB4HB/PVA core-shell nanofibers to produce neobone upon coaxial bioelectrospinning simultaneously with differentiated human bone marrow mesenchymal stem cells(h BMSCs). Methods: The nanofibers scaffolds were prepared by coaxial bio-electrospinning P3HB4HB/PVA-h BMSCs in vitro with or without osteogenic media for 14 days. The complex of P3HB4HB/PVA-h BMSCs were assessed by using SEM, fluorescence staining and DAPI staining. The induces and noninduced complexes of P3HB4HB/PVA-h BMSCs were implanted into the subcutaneous in the back of nude mics. The complexes of P3HB4HB/PVA-h BMSCs were terminated in vivo, assessed by using HE staining, alizarin red staining, von kossa staining, masson staining, collagentype I staining at 8, 16 weeks after they were impanted in vivo. Results: Scanning electron microscopy showed that the nanofibrous were continuous and uniform, cell mass and uniform distribution of the nanofibers in each layer,differentiated cells produced abundant extracellular matrices with increasing culture time. No cytotoxicity was observed by the fluorescence staining and DAPI staining. After 14 days of in vitro culture, the differentiated P3HB4HB/PVA-h BMSCs nanofibers scaffold were implanted into the subcutaneous layer nude mice for 8 or 16 weeks, non-differentiated P3HB4HB/PVA-h BMSCs nanofibers scaffold were implanted as the control group. The differentiated P3HB4HB/PVA-h BMSCs implants formed bone-like tissue after 16 weeks of implantation, and stained positive for alizarin red staining, von kossa staining, masson staining, collagentype I staining. Conclusion: This study demonstrated that coaxial bio-electrospinning of cells and materials, not only can make the scaffolds complication and nanocrystallization, but also can realize the precise cultivation of cells,is a suitable material for bone tissue engineering.