The Effect Of Fullerene Derivatives On The Osteogenic Differentiation Of Mouse Mesenchymal Stem Cells Under Different Culture Conditions

ObjectiveTo investigate how fullerene derivatives such as fullerene tris-acid(FTA)and fullerol affect osteogenic differentiation of mouse Bone Marrow Mesenchymal Stem Cells(BMSCs).Methods 1.Cultured mouse bone marrow mesenchymal stem cells(BMSCs)which is also called D1 cell lines.2.Treatment for cells.Part I: The effect of fullerenes derivatives on osteogenic differentiation of mouse BMSCs under normal cell culture condition.Blank control was set as DMEM culture medium supplemented with 10% fetal bovine serum(FBS)and 100?g/ml hybrid antibiotics(penicillin and streptomycin,P/S).On the basis of blank control,added 10 mm ?-glycerophosphate(?-GP)as the experimental control.Experimental group was set as experimental control added with different concentration of fullerene derivatives FTA or fullerol.The concentration gradient of FTA was set to 0.25?M,0.5?M,1.0?M,2.0?M,the concentration gradient of fullerol was set to 0.33?M,1.1?M 10?M,3.3?M.Part II: The effect of fullerenes derivatives on osteogenic differentiation of mouse BMSCs under exogenous oxidative stimulation.Blank control was set as DMEM+FBS+P/S.On the basis of blank control,treated with hydrogen peroxide(H2O2)20 mM for 2 hours as the experimental control.Experimental group was set as experimental control pretreated with different concentrations of fullerene derivatives FTA or fullerol for 2 hours.The concentration gradient of FTA was set to 0.25?M,0.5?M,1.0?M,2.0?M,the concentration gradient of fullerol was set to 0.33?M,1.1?M 10?M,3.3?M.Subsequently,each group was replaced with an osteoinductive medium(DMEM+FBS+P/S+?-GP).Part III: The effect of fullerenes derivatives on osteogenic differentiation of mouse BMSCs under serum starvation.Blank control was set as DMEM+FBS+P/S.On the basis of blank control,removed the FBS from culture medium as the experimental control.Experimental group was set as experimental control pretreated with different concentrations of fullerene derivatives FTA or fullerol for 2 hours.The concentration gradient of FTA was set to 0.25?M,0.5?M,1.0?M,2.0?M,the concentration gradient of fullerol was set to 0.33?M,1.1?M 10?M,3.3?M.Subsequently,each group was replaced with an osteoinductive medium(DMEM+FBS+P/S+?-GP).3.Test and evaluation.At different time of cell culture,alkaline phosphatase(ALP)content was determined and the cell mineralization was evaluated by alizarin red staining.LDH and WST-1 colorimetric kits were used for cell toxicity and proliferative assays respectively.Cell apoptosis and ROS level were measured by flow cytometry.The protein expression level were measured by Western Blotting.Results 1.Normal cell culture conditionCompared with the experimental control,the application of FTA or fullerol enhanced the ALP activity(P < 0.05)with concentration dependence.The number of BMSCs mineralized nodules was increased by the FTA,especially in the high concentration of 2.0mM(P < 0.05),while the effect by fullerol was limited.After osteogenic induction,the ROS level increased greatly in the first day compared with the blank control(normal medium without osteogenic inducer),and the addition of FTA reduced ROS level.As for protein expression,western blotting showed that cells treated with an osteogenic medium had a higher expression of Runx2,SOD2 and p-JNK compared with the blank control,however inhibited p-Akt expression.Furthermore,treated with different dose of FTA or fullerol on this basis rasied Runx2,p-JNK,p-Akt levels respectively,and the effect of low concentration for the FTA was more apparent.2.Exogenous oxidative stimulation culture conditionAfter the FTA treatment,the BMSCs mineralized nodules were obviously increased(P < 0.05),which was more significant in the middle and low concentration of 0.25?M and 0.5?M,and fullerol had little effect on mineralization.Additionally,there was no obvious difference for the ALP activity treated with FTA or fullerol.Compared with the blank control(without H2O2),treated with H2O2 inhibited cell proliferation apparently,and it was reversed by adding the FTA in some degree(P < 0.05),while fullerol didn't show much effect.Besides,H2O2 increased cells mortality evidently,and FTA or fullerol reduced the cell death rate in low or high concentrations respectively.Furthermore,it was suggested that cell death was involved with apoptosis caused by oxide stimulation.As for protein expression,western blotting showed that cells treated with oxidative stimulation had a higher expression of p-Akt,SOD2 and FoxO1 compared with the blank control,and the expression was further increased under the action of FTA or fullerol,and there was little difference between the experimental group and experimental control about the Runx2 level.3.Serum starvation culture conditionThe results of cell Fas protein detection and PI staining showed that both FTA and fullerol inhibited the cell apoptosis unregulated by serum starvation.However,the mineralization test,cell proliferation and toxicity assays results showed that there was no significant difference in the application of FTA or fullerol compared with the control group.Conclusions1.Under the normal culture condition,antioxidant FTA promote mouse bone marrow mesenchymal stem cells(D1 cells)osteogenetic differentiation,embodied in the mineralization,the activity of ALP and osteogenesis specific transcription factor Runx2 protein levels.2.In the state of exogenous oxidative stimulation,FTA reverse the disadvantage factors,promote D1 cells proliferation and osteogenic differentiation,and inhibit cell apoptosis caused by oxidative stress.3.The FTA affect the signaling pathway of p-JNK,p-Akt and FoxO1 by reducing the cell ROS level,and increase the expression of SOD2 to promote the osteogenic differentiation of D1 cells.