The Effect Of Cyclic Tensile Strain In Growth Plate Chondrocytes And Possible Mechanisms Of Mechanotransduction

Objective1.To study the3D culture and co-culture of rat growth plate chondrocytes and bone marrow mesenychemal stem cells, then discuss the advantages and disadvantages of them as seed cells for tissue engineering.2.To investigate the effect of cyclic tensile strain on the proliferation of rat growth plate chondrocytes and to explore whether ERK and YAP protein were involved in this process.3.To observe the role of FAK, cytoskeleton F-actin and RhoA signaling in the mechanotransduction mechanisms of rat growth plate chondrocytes responding to tensile strain.Method1.Rat growth plate chondrocytes and bone marrow mesenchymal stem cells were isolated and cultured in vitro. Light microscope was used to observe the cell morphology. Rat bone marrow mesenchymal stem cells were induced to three lineages and examine the ability of differentiation. Both cells were cultivated in3D and co-culture method. The experiment was divided to three groups:growth plate chondrocytes monoculture, bone marrow mesenchymal stem cells monoculture and the two cells co-culture. The expression of the chondrogenic genes were examined by Real-time PCR. The expression of extracellular matrix of three groups were detected by immunohistochemistry.2.By using four point bending device, rat growth plate chondrocytes were subjected to cyclic tensile strain. The expression of YAP,ERK,PCNA and CyclinD1protein were examined by western blot. The expression of C-Fos and CTGF mRNA were examined by Real-time PCR. When the activation of ERK or YAP was blocked, western blot and flow cytometry were used to examine whether the effect of tensile strain in chondrocytes was changed.3.Rat growth plate chondrocytes were subjected to cyclic tensile strain. The activation of FAK and RhoA protein were examined by western blot. The effect of tensile strain in cytoskeleton F-actin was observed by immunofluorescence. When the activation of FAK or RhoA was blocked, western blot was used to examine the change of the activation of YAP and ERK protein by cyclic tensile strain.Result1.The morphology of rat growth plate chondrocytes were triangular or short spindle, while rat bone marrow mesenchymal stem cells were spindle. Bone marrow mesenchymal stem cells could differentiate into three lineages. Despite the chondrogenic differentiation of co-culture group was better than the other two groups at early stage, rat growth plate chondrocytes monoculture group was strongest in chondrogenic differentiation at late stage.2.When rat growth plate chondrocytes were subjected to cyclic tensile strain (at3000μstrain,0.5Hz)for1hour, the activation of YAP and ERK protein were highest, and the same to the expression of C-Fos and CTGF mRNA. Proper tensile strain could induce the proliferation of chondrocytes and change the cell cycle. If the activation of ERK or YAP protein was inhibited, the effect of tensile strain was blocked.3.Cyclic tensile strain could activate the FAK and RhoA protein in rat growth plate chondrocytes and polymerize the cytoskeleton F-actin. If the cells were treated by PF573228, C3toxin or cytochalasin D,the activation of YAP and ERK were blocked.Conclusion:1.Rat growth plate chondrocytes and bone marrow mesenchymal stem cells both have advantages and disadvantages to be seed cells for tissue engineering.3D and co-culture were benefit for the development of growth plate tissue engineering.2.Cyclic tensile strain could induce the proliferation of rat growth plate chondrocytes, and the activation of ERK and YAP protein were involved in the process.3.Cyclic tensile strain could affect the expression of ERK and YAP protein through FAK, RhoA and cytoskeleton F-actin.