A Preliminary Study Of Fluorescent Carbon Dots Tracing And Promoting Osteogenic Differentiation Of Rat BMSCs

Purpose:Rat bone marrow mesenchymal stem cells（BMSCs）were isolated and cultured in vitro,and labeled by long-wave carbon dots（CDs）to investigate its ability of tracing cells and effects on cell osteogenic differentiation.Methods:1.BMSCs of 3-week-old male SD rats（provided by Experimental Animal Center of Nanjing Medical University）were isolated and cultured.Cell morphology was observed by optical microscope.Cell phenotype were identified by flow cytometry.Alkaline phosphatase（ALP）staining and alizarin red staining were performed to detect the osteogenic differentiation potential of the isolated cells.Adipogenic differentiation potential was identified by oil red O staining.2.CDs were characterized by transmission electron microscope（TEM）and Fourier transform infrared spectroscopy（FT-IR）.The effects of CDs on cell activity of BMSCs were measured by CCK-8 assay at 1,3,5 and 7 days respectively.Flow cytometry was applied to detect the concentration dependence of the efficiency of CDs labeling cells.A variety of endocytosis inhibitors and culturing at 4℃low temperature were used to explore the pathways of cells uptaking CDs.Laser scanning confocal microscopy was used to observe the imaging characteristics of BMSCs.The intracellular distribution of CDs was confirmed by fluorescence co-localization with green lysosome fluorescent probe,and the labeled cells were traced for a long time.The early osteogenic differentiation level of BMSCs was determined by ALP activity at 4 and 7 days after osteogenic induction.The mineralization level of BMSCs extracellular matrix was detected by alizarin red staining at 21 days after osteogenic induction.The relative expression levels of osteogenic related genes OPN,OCN,Runx2 and Osterix were detected by RT-PCR at 7 and 14 days after osteogenic induction.Results:1.Rat BMSCs were successfully isolated and cultured in vitro,and identified by flow cytometry,ALP,alizarin red and oil red O staining.2.TEM showed that CDs were spherical particles and the average particle size was 2.17 nm.FT-IR analysis showed that the surface of CDs was rich in hydroxyl and amino groups.CCK-8 results showed that compared with the control group,the cell activities of 50,100μg/m L CDs groups significantly increased at the 1^st day（P<0.05）and the cell activity of 300μg/m L CDs group decreased（P<0.05）,but was still higher than 80%.Flow cytometry results showed that the labeling efficiency increased with the increase of CDs concentration.Red fluorescence of BMSCs was observed under the excitation of 561 nm by laser scanning confocal microscopy after co-culture with CDs.The red fluorescence of CDs partly located in cytoplasm and partly overlapped with the green fluorescence of lysosome,but did not enter the nucleus.Fluorescence could still be seen by laser scanning confocal microscopy after 9 days of co-culture.The uptake amounts of CDs by BMSCs which inhibited at 4℃were significantly decreased（P<0.05）.The ALP activity of CDs group was higher than that of blank control group（P<0.05）.The results of alizarin red staining showed that the extracellular matrix mineralization level of CDs group was higher than that of control group.Reverse transcriptase-polymerase chain reaction（RT-PCR）results showed that the relative expression levels of osteogenic related genes OPN,OCN,Runx2 and Osterix of CDs group were higher than those in control group（P<0.05）.Conclusion:CDs show good biological safety.CDs have the potential to trace BMSCs and promote the osteogenic differentiation of rat BMSCs.