Abstract: Objective To investigate the feasibility of CM-DiI on long-term tracing the biological change of bone mesenchymal stem cells(BMSCs) during osteogenesis of tissue engineered bone(TEB) in vivo,and to offer an alternative labeling method for tracing seed cells in vivo in future.Methods After BMSCs were separated from bone marrow in Beagle dogs and osteo-induced to the second generation(P2),the cells were labeled with the fluorescent dye CM-DiI.The morphologic change of BMSCs after labeling was observed by fluorescence convert microscope.The proliferative ability was tested by MTT colorimetric between the labeling and un-labeling groups.The expression of Collagen I,BMP-2,BGLAP,and SPARC were detected through RT-PCR between the two groups.Lastly,CM-DiI-labeling BMSCs and beta-TCP were co-cultured into TEB which was implanted subcutaneously into Beagle dogs.Eight weeks later,the implant was harvested and the biological change of BMSCs was observed by fluorescence convert microscope.At the same time,the ectopic osteogenesis of the TEB was tested by HE staining.Results Shortly after being labeled with CM-DiI,ringlike red fluorescence was present in the cytoplasm of BMSCs.48 hours later,the fluorescence was enhanced and was not weaken during 72 hours.The morphology of labeled BMSCs was consistent with unlabeled ones.There was no significant difference in the proliferative ability between the labeled and unlabeled groups.The expression of Col-Ⅰ,BMP-2,BGLAP,SPARC revealed that the CM-DiI labeled BMSCs were induced into osteoblasts successfully either.After seeding into β-TCP,the proliferation of labeled BMSCs was normal and abundant extracellular matrix(ECM) was observed in the pores of the ceramic by scanning electron microscope(SEM).After harvesting the composite of labeled cells and ceramic 8 weeks later,the cells with red fluorescence were observed by fluorescent microscope.HE staining revealed deposited osteoid in the pores of ceramic.Conclusion CM-DiI has no obvious impact on the growth,proliferation and osteogenic differentiation of BMSCs.The BMSCs in the composite of TEB could be traced as long as 8 weeks.It suggests a feasible method in monitoring of cells in vivo.

Key words: Tissue engineered bone, Bone mesenchymal stem cells, Ectopic osteogenesis, CM-DiI