A Triple-model Imaging Study For In Vivo Tracking The Location Viability And Osteogenic Differentiation Of Transplanted Stem Cells In Bone Regeneration

Objects:Developing a triple-labeling model imaging strategy to in situ track the location,viability and osteogenic differentiation of transplanted stem cells in vivo by combining the Ag2S QDs-based near-infrared fluorescence imaging and,red firefly luciferase-based(RfLuc)bioluminescence imaging and Gaussia luciferase-based(GLuc)bioluminescence imaging.Methods:The RfLuc and GLuc reporter-genes were cloned into the lentiviral vector(pLVX-CMV-RLuc-Colla-GLuc).Then the pLVX-CMV-RLuc-Colla-GLuc and the lentiviral backbone vector were homologous recombined in 293T cells.The lentivirus were produced using 293T cells and purified.Then,a triple-labeling strategy was studied to label hMSCs with Ag2S QDs,RfLuc and GLuc.After that,systematical analyses were performed to test the biocompability of the triple-labeling strategy.Mice with a calvarial defect were induced by medical orthopaedics drill.The triple-labeled hMSCs were cultured in collagen scaffold and transplanted to the calvarial defect site.The BLI images and NIR-Ⅱ fluorescence images were obtained after cell transplantation.The therapeutic potential of triple-labeled hMSCs for bone regeneration was performed by CT and histology assay.Results:A triple-labeling strategy was successfully developed to label hMSCs with RfLuc,GLuc and Ag2S QDs.A systematical cytotoxicity study suggested the high biocompability of the triple-labeling strategy with no adverse effects on cell viability,proliferation and osteogenic differentiation.Then,a multi-model imaging was developed to in vivo visualize the location,viability and osteogenic differentiation of transplanted stem cells by combining the exogeneous near-infrared fluorescence in the second window(NIR-Ⅱ)and edogeneous bioluminescence imaging(BLI).It was found that the the triple-labeled hMSCs could be imaged by both IVIS Lumina Ⅱsystem and NIR in vivo imaging system.The dynamic translocation,viability and survival and differentiation of transplanted cells were systematically imaged and semi-quantified by using the combined NIR-II PL imaging and BLI method in mice with acute calvarial defect.In combination with the bone regeneration assay,the results suggested that the improved cell survival and osteogenic differentiation of transplanted hMSCs.Conclusions:We have developed a combined BLI/NIR-Ⅱ strategy for dynamic monitoring cell translocation,viability and osteogenic differentiation in vivo with desirable spatial resolution and sensitivity.With the biocompatible nature of the triple-label system,we expect that it will have great promises in a broad range of biomedicine and stem cell studies,such as imaging-guided cell therapeutics,drug screening,and so on.