| Background According to the recent study,increasing the amount of bone mesenchymal stem cells(BMSCs)and enhancing the regulation of bone Morphogenetic Proteins(BMPs)play a key role in bone regeneration and repair.Gene transfection can achieve an perfect combination of the two.However,currently,before the clinical application of gene transfection on bone regeneration and repair,we should solve the problem of poor safety,low gene transfection efficiency and invisible and uncontrollable transfection process.Objective By Integrating osteoblast seed cells---Bone Marrow Mesenchymal Stem Cells(BMSCS)and osteoclast cytokine(recombinant human Bone Morphogenetic Protein-2,rhBMP-2)through gene transfection,this study put forward a kind of biosynthesis nanobubbles called Gas Vesicles(GVs)with advantages of the biocompatibility and small particle size and ultrasound imaging capability.They can also be the perfect carrier for rhBMP-2 gene.In this research,GVs loaded with rhBMP-2 gene was endocytosed by BMSCs.Intracellular cavitation effect occurs in the cell mediated by ultrasound will lead to an efficient,safe,visible nuclear gene delivery methods.In addition,from the angle of ultrasound imaging and gene transfection expression ability,the potential of visualizing regulation of rhBMP-2 gene transfection mediated by ultrasound to promote bone repair in vivo was preliminarily investigated.This study will provide a new approach for the regenerative treatment of stem cell tracking and gene transfection,and provide a new strategy for the comprehensive treatment of bone regeneration.Methods 1.Modified the traditional method by passaging and reducing the energy of centrifugation to prepare GVs with high efficiency.GVs was cationized by Polyetherimide(PEI)to construct GVs-PEI.Morphological characterization was analyzed by transmission electron microscopy(TEM).Particle size and zeta potential were measured by dynamic light scattering(DLS).Ultrasound imaging ability and stability were tested by ultrasound imaging system.2.GVs@BMSCs were prepared by co-incubation,and the phagocytosis ability of GVs-PEI by BMSCs and its stability in cells were detected by fluorescence microscope.The proliferation activity of stem cells after preparation for 24-72h was determined by Cell Counting Kit-8(CCK-8)test.GVs@BMSCs ultrasound imaging was operated in agar phantom and in rat quadriceps to detect the potential of ultrasound enhanced imaging in vitro and in vivo.3.? rhBMP2-GVs@BMSCs gene transfection system were synthetized by co-incubation.?GVs fluorescence staining and Calcein-AM/PI staining were used to verify the mechanism of gene delivery of intracellular cavitation from the perspective of GVs nucleus entry efficiency and nuclear membrane opening effect.?Optimal ultrasound parameters of in vitro transfection was explored by comparing the expression rate and cell viability of EGFP transfected with different acoustic intensity ultrasound.? EGFP expression was observed under a fluorescence microscope and rhBMP-2 secretion was quantitatively analyzed by ELISA after transfection.?Normal BMSCs were cultured by cell culture supernatant of different group.The effectiveness of BMSCs in promoting osteogenic differentiation was verified by ALP and calcium nodule formation.Result 1.A higher concentration of GVs was prepared by modified method.TEM image showed that GVs was a spindle-shaped balloon structure with uniform shape and size.GVs was cationized to synthetize GVs-PEI and DLS test results showed that the average diameter was(383.6±11.55)nm and the zeta potential was(18.5±2.20)mV.Ultrasound imaging results showed that with the increase of GVs-PEI concentration in vitro,the ultrasound signal intensity was also enhanced,and the optimal imaging concentration was OD500=1.0,which can last for 96h2.We constructed GVs@BMSCs successfully.Fluorescence image results revealed that GVs-PEI showed a large amount of red specular fluorescence in the cytoplasm of BMSCs,and could be stable for 4 days.When the incubation concentration was OD500=0.5-1.0,the endocytic marking of GVs did not affect the proliferation activity of BMSCs.Ultrasound imaging results showed that both in vivo and in vitro GVs@BMSCs showed significant enhancement of ultrasound imaging,and the signal intensity difference was statistically significant compared with that of the BMSCs group alone,and the imaging effect can last for at least 6 days(P<0.001)3.? rhBMP2-GVs@BMSCs gene transfection system was successfully constructed.? Fluorescence staining was used to verify the gene delivery mechanism of intracellular cavitation effect mediated by ultrasound.GVs fluorescence staining results showed that GVs red spot fluorescence overlapped with nucleus' blue fluorescence in BMSCs after ultrasound irradiation,stating that GVs entered the nucleus successfully.Calcein/PI staining results showed that BMSCs showed green fluorescence in cytoplasm and red fluorescence in nucleus after ultrasound irradiation,explaining that the permeability of cell nuclear membrane was improved.? When the ultrasound parameter was 1 W/cm2,1MHz,duty cycle 20%,and 2min,EGFP expression was high and no significant effect on cell viability was observed(P>0.05).Therefore,it was the optimal ultrasound parameter to stimulate the intracellular cavitation effect for transfection.? The expression level and fluorescence intensity of EGFP in the US+rhBMP2-GVs@BMSCs group were significantly higher than those in the other control groups,and the expression lasted for 14 days.ELISA test showed that the concentration of rhBMP-2 protein was significantly higher in US+rhBMP2-GVs@BMSCs group than that of the normal BMSCs group(P<0.001),and the secretion lasted for 21 days.? The expression of ALP and the generation of calcium nodules in US+rhBMP2-GVs@BMSCs group were significantly higher than those in the control group and the osteogenic differentiation of BMSCs were promotedConclusion The marking of GVs-PEI mediated by endocytosis has realized a safe,stable and long-term imaging tracking of BMSCs under ultrasound,which provides a new scheme for the tracking of stem cells in vivo.Ultrasound-mediated rhBMP2-GVs@BMSCs gene transfection with intracellular cavitation effect can significantly improve the permeability of cell nuclear membrane,and achieve a safe and efficient nuclear delivery and expression of rhBMP-2.The secretion of rhBMP-2 is sustained and stable,so as to adjust the internal environment and promote osteogenic differentiation of BMSCs.Moreover,this system is expected to achieve an in vivo ultrasound visual able and controllable BMSCs gene transfection method for bone regeneration and repair. |
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