MRI Tracing And Magnetic Targeting Performance Of GLcN-PLL-USPION In Bone Tissue Engineering

Background:Bone marrow mesenchymal stem cells(BMSCs)are currently the most ideal seed cells for bone tissue engineering and stem cell therapy.Molecular imaging techniques such as magnetic resonance imaging(MRI)are commonly used for live tracing after BMSC implantation in vivo.MRI tracer needs to label BMSC with superparamagnetic iron oxide(SPION)or ultra-small superparamagnetic iron oxide(USPION),but SPION and USPION have defects such as low cell uptake rate and nanoparticle agglomeration.To overcome these shortcomings,the current common approach is to modify SPION and USPION.This project intends to design USPION labeled BMSCs with double modification of D-glucosamine(GLcN)and poly-L-lysine(PLL)to achieve in vivo tracking.At the same time,the magnetic targeting performance after labeling BMSC was explored.Objective:The purpose of this study is to:? modify the USPION by two transfection reagents,GLcN and PLL,to enhance cell uptake rate and dispersibility;? to evaluate the labeling effects after modification and its effect on BMSC properties,and compare the advantages and disadvantages between different modifications;?test the in vitro and in vivo duration of GLcN-PLL-USPION labeled BMSC,and investigate the MRI tracer performance in vivo;? test the magnetic targeting ability of GLcN-PLL-USPION under magnetic field to explore the potential and application prospects as magnetic targeting materials.Method:A double modified GLcN-PLL-USPION was synthesized using USPION containing a PMAA coating.The optimal cell marker concentration was tested and the marker effect was evaluated by calculating the Prussian staining area,cell labeling rate,and intracellular iron content.Transmission electron microscopy was used to observe whether USPION was taken up by BMSC and the distribution in the cells.By comparing the labeling efficiency and intracellular iron content of different modified USPION cells,it was evaluated whether the double modification has a significant advantage over the single modification.The duration of labeling in an in vitro environment was measured after labeling BMSC.The GLcN-PLL-USPION-labeled BMSC was loaded into a thiol-modified hydroxyapatite hydrogel and implanted into SD rats to evaluate in vivo imaging and in vivo labeling duration.Finally,the migrating and invasive ability of GLcN-PLL-USPIO labeled BMSC was tested by static magnetic field to evaluate the magnetic targeting effect.Results:50 ?g/ml was the optimal concentration for GLcN-PLL-USPION,with a labeling efficiency of more than 99%and a cell iron content of 18.08±1.14 pg/cell.At this concentration,BMSC labeling did not affect proliferation performance and triline differentiation potential.Transmission electron microscopy confirmed that GlcN-PLL-USPION was present in the cytoplasm after being taken up by the cells.Comparing the effects of different modified markers,it was found that the double modification was significantly better than the single modification,demonstrating that the double modification helps to increase the dispersibility and increase the cell uptake rate.In vitro,the presence of labeled cells was observed after 17 days after GLCN-PLL-USPION labeled BMSC.In vivo experiments,labeled cells were implanted in CS-TBA/HA/?-GP hydrogel and implanted into rats.The MRI imaging effect was significant and the imaging duration was maintained for more than 13 days.In addition,GLcN-PLL-USPION performs targeted movement under static magnetic field,demonstrating the magnetic targeting potential of GLcN-PLL-USPION labeled BMSC.Conclusion:The double-modified GLcN-PLL-USPION can achieve a labeling rate of more than 99%for BMSC,an in vitro labeling duration of about 17 days,and a labeling time of more than 13 days in vivo.MRI imaging is effective,which is very suitable as a Tracer of BMSC in bone tissue engineering.In addition,GLcN-PLL-USPION labeled BMSC can be effectively pulled under the action of static magnetic field,which proves that GLcN-PLL-USPION has magnetic targeting ability in addition to MRI imaging effect.