Effects Of Uniaxial Cyclic Stretching On Growth And Differentiation Of Goat Bone Marrow Mesenchymal Stem Cells

Objective: Bone marrow mesenchymal stem cells(BMSCs)are considered to be important sources of tissue engineered seed cells due to their ability to diversify differentiation.How to induce their differentiation has been a major problem in tissue engineering.Biomechanical signals can affect the differentiation of BMSCs,but the stress loading type,intensity,frequency and duration are different.BMSCs differentiation direction and degree are different.In the process of tensile stress,cell-extracellular matrix(ECM)interactions,cytoskeleton et al have alteration and functional transformation.In this study,the changes of cytoskeleton and cell function of goat bone marrow mesenchymal stem cells(gBMSCs)were detected by uniaxial cyclic tensile stimulation with different strength.Methods:1.In vitro isolation,culture of gBMSCs,cell morphology observation.2.The effects of 10%,15% and 20% tensile stress on the cell morphology and cell proliferation of gBMSCs were measured.3.Toluidine blue and Sirius red staining were used to identify the gBMSCs with different tensile strength and the related software were used for cell counting analysis.4.Immunohistochemical staining of type Ⅰ collagen was carried out to identify the gBMSCs with different tensile strength and the related software for cell count analysis.5.Immunofluorescence staining was used to detect the effect of different uniaxial tensile stress on cytoskeleton of gBMSCs.6.Real-time fluorescence quantitative reverse transcription polymerase chain reaction(RTqPCR)was used to observe the expression of specific genes of bone marrow mesenchymal stem cells under different tensile stress.Results:1.Load tensile strength at different intensities,but 15% and 20% of the intensity,the cells were significantly elongated and perpendicular to the tensile stress in the direction Y axis;2.MTT results showed proliferation of gBMSCs were observed under 10% and 15% intensity of the induced group.Flow cytometry showed that the proliferative activity of 10% and 20% cells was significantly enhanced.3.The contents of Glycosaminoglycan(GAGs)in gBMSCswere significantly increased by 10% and 15% group.The fibers were tightly arranged in red stained by Sirius red,and the content of collagen was increased by 10% and 20% group.4.Actin fluorescence immunostaining displayed the cytoskeleton was intact both induction group and negative control group,and the skeletal fiber was longer in the induction group,but the fluorescence intensity of the induced group was significantly lower than that of the negative control group.The anti-Vinculin immunofluorescence staining showed the fluorescence intensity of 10% group was obviously higher than that of the negative control group and the other two groups.5.RT-qPCR showed the relative expression of gBMSCs related gene(CollagenⅠ,collagenⅡ,GAGs,Vinculin)in induced group was significantly higher than that in the control group(P <0.01).In contrast,the relative expression level of α-SMA decreased to 35% in the control group,15% group decreased to 29% in the control group,and 20% group decreased to 20% in the control group.Such as ColⅠ: 20% group was apparently higher than the control group and the other two groups;Col Ⅱ: 10%,20% group was significantly higher than the control group and other experimental groups;GAGs: 10% and 15% group were obviously higher than those in the control group and other experimental groups;Vinculin: 10% group was apparently higher than the control group and other experimental groups.Conclusions:1.The tensile strength of 20% has a certain role in promoting gBMSCs differentiation to fibroblastoid cell;2.The uniaxial cyclic tensile stress can be induced to differentiate the bone marrow mesenchymal stem cells into fibrouscartilage-like cells.