Ultrasound Biological Effects Upregulate The Stemness And Proliferation Of Bone Marrow Mesenchymal Stem Cells

ObjectiveTo explore the regulation of different types of ultrasonic biological effects on stemness of MSCS,and to screen the appropriate ultrasonic parameters for up-regulating the expression of stemness gene and promoting proliferation of MSCS.MethodsMSCSs were extracted from the femoral and tibial bone marrow of SD rats aged 3-4 weeks and cultured by adherent method until the third generation and then inoculated into 6-well plates.The US treatment parameters were frequency1 MHz,pulse repetition frequency(PRF)100 Hz,duty cycle 1%,and total treatment time 5 min.The peak negative pressure is adjusted to 0.5 MPa,1MPa and 1.5 MPa successively.According to whether microbubbles were added,the experimental groups were divided into UM groups(MSC+0.5 MPa US+MBs,MSC+1.0 MPa US+MBs,MSC+1.5 MPa US+MBs);LU groups(MSC+0.5MPa US,MSC+1.0 MPa US,MSC+1.5 MPa US),and the control group was connected to the ultrasonic transducer for 5 min without stimulation.0.01 m L liposomal MBs were diluted in 1 m L cell culture medium,mixed and added to each UM group,and 1 m L of the same cell culture medium was added to the LU group.After irradiation,the Apoptosis Detection kit was used to detect the apoptosis of mesenchymal stem cells in UM group and LU group.The Cell Counting kit(CCK8)was used to detect the proliferation of MSCs at different effects and time points(12 h,24 h,36 h).A Enzyme-linked Immunosorbent assay(ELISA)was used to determine whether the UM group was more effective than LU group in promoting the expression of stemness genes(NANOG and OCT-4).Reverse transcription quantitative PCR(RT-PCR)and Western blotting were used to detect the expression levels of stemness genes(OCT-4 and SOX-2)and their proteins,so as to determine the optimal parameter setting of ultrasound.ResultsApoptosis assay suggested that mechanical and cavitational treatments did not increase MSC apoptosis(P>0.05).Proliferation assay showed that 24 h after treatment MSC proliferation was most significantly increased in the 1.5MPa UM group(P<0.01).The expression of stemness genes was significantly increased in both the 1.5 MPa LU group and UM group compared with the control group,but the highest expression was in the 1.5 MPa UM group(P<0.01).ConclusionsThe cavitational effect at 1.5 MPa peak negative pressure significantly upregulated the expression of MSC stemness gene and promoted MSC proliferation at 24 h after irradiation.