| Objective:(1) the lentivirus bone marrow mesenchymal stem cells(BMSCs) and verify that the increase of BMSCs express CXCR4 after transfected;(2) detection of the directed chemotactic ability of CXCR4+-BMSCs to stromal cell-derived factor l(SDF-1);(3)Construction of SDF-1/collagen type I/chitosan composite membrane, detection of the characterization of membrane, swelling ratio, biocompatibility, controlled slow release SDF-1;(4) the tibial defect model of goat constructed by the SDF-1/collagen type I/chitosan composite membrane complex in bone tissue engineering, observing CXCR4+-BMSCs and BMP-2 apply to promote the large bone defect repair effect for final treatment of large bone defects may provide a treatment on.Methods:(1) bone marrow adherent cultured goat BMSCs and through the osteogenic,adipogenic, chondrogenic multi-inducing and identified by the corresponding staining;observing cell morphology and activity from a morphological point of view;using the CXCR4 gene lentiviral vector transfect BMSCs, from the fluorescence expression,protein expression to verify transfection successfully;(2)CXCR4+-BMSCs to SDF-1chemokine ability was stronger than BMSCs, which was verified by Transwell experiment;(3) the use of freeze-drying to prepare of SDF-1/collagen type I/ chitosan composite membrane, the size and shape of the aperture and test swelling ratio by scanning electron microscopy.imitated the effects of biodegradation of film by lysozyme, using an ELISA kit for each set of test liquid sustained release SDF-1concentrations were detected; detected by MTS assay for cell proliferation and differentiation;(4) building the defect model of large bone of goat tibia by using the intervention microcirculation system, and use this animal model, giving BMSCs treatment and BMP to this model, observing the goat and using radiographic to obverse the change of tibia repair.(5) using the SPSS 19.0 statistical software to analyze the data, which are expressed as mean±standard deviation, student- newmankeuls test was used to statistical analysis between groups. Differences between thegroups was analysed by ANOVA statistics and it is significant when P<0.05.Results:(1) the primary and subculture BMSCs stained by LIVE/DEAD cell imaging kit basically showing good activity of green fluorescence intensity, DAPI staining showed good nuclear morphology without obvious apoptosis, the expression of CXCR4 in BMSCs turn significantly higher levels than before gene transfection;(2)Transwell experiments showed the chemotaxis capacity of CXCR4+-BMSCs to SDF-l were higher significantly than BMSCs(p<0.05);(3) SDF-1/collagen type I/ chitosan composite membrane was successfully prepared,The composite membrane was porous spongy, swelling rate of 98%, an average pore diameter was(131±12) um; the composite membrane could be degraded in PBS containing lysozyme, SDF-1 could be slow and sustained released; BMSCs could maintain cell proliferation in cultured on composite membrane, the proliferation of dividing cells were observed through electron microscopy and spectroscopy scans;(4) successfully constructed intervention microcirculatory system repair large goat tibia bone defect model, the functional of goat recovery well, the upper and lower callus connected closely and tissue engineering bone resorption were visible by imaging.Conclusion:(1) high viral tite and overexpression of CXCR4 lentiviral vectors can transfected BMSCs which culture by the whole bone marrow adherent method, and the chemotaxis capacity of CXCR4+-BMSCs to SDF-l were improved higher significantly after gene transfected;(2) SDF-1/collagen type I /chitosan composite membrane could degradate in vitro and release SDF-1 with good biocompatibility.(3)intervention microcirculatory system combines slow release drug delivery membrane systems and tissue engineering in goat tibia bone repair large bone defects could achieve good results, it is a new method for repairing bone defect. |
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