Effect of equiaxial tensile strain in early differentiation of mesenchymal stem cells into cartilage cells

Abstract

To observe the effect of cyclic equiaxial tensile strain in the early differentiation of bone marrow mesenchymal stem cells(BMSCs) into cartilage in mouse under conditions of two-dimensional culture, and to investigate the mechanism of cyclic equiaxial tensile strain in early chondrogenic differentiation. Sixteen KM mouse aged 4 weeks were selected, male and female unlimited, with an average weight of 19.5 g (17 to 21 g). After extracting and isolating the BMSCs from KM mouse, then subculture the BMSCs to the 3rd generation. Seed the cells in the biological plate(BioFlex). According to experimental design, the cells were divided into 6 groups, blank group: ordinary culture medium was cultured for 8 days without isometric cyclic tensile strain stimulation. Control group: chondrogenic differentiation medium was used to culture for 8 days without isometric cyclic tensile strain stimulation. Experimental group: the experimental group was divided into 4 groups, all of which were cultured with chondrogenic differentiation medium for 8 days. During which isometric cyclic tensile strain stimulation was given for 1, 3, 5 and 7 days respectively. At the 8th day, all the cells were collected, the expression of the Sox9, Col-II and ROCK 1 signaling pathway-related molecules was analyzed by RT-PCR. Cells in each group were extracted, and the efficiency of cell proliferation in each group was detected by CCK-8. Glycosaminoglycan content in medium changed last was detected using ELISA. Pericellular matrix was observed by Safranin O staining and Alcian Blue staining. Normal measurement data using mean±standard deviation compared between the blank group and control group using paired t-test, compared between the experimental group and relative group using single factor analysis of variance. (1)After 8 days of culture, compared with the control group, the relative expression of Sox 9 and Col-II mRNA in the experimental group increased gradually with the increase of loading time(P<0.05), while the relative expression of ROCK1 mRNA decreased(P<0.05). Compared with the blank group, the relative expression of ROCK1 mRNA in experimental group and control group increased (P<0.05). (2)With the increase of loading time, the experimental group showed a trend of decreasing at first and then increasing, but compared with the blank group and the control group, the control group decreased significantly. (3)Glycosaminoglycan content in the medium changed last was detected by ELISA. The glycosaminoglycans in the experimental group increased gradually, and the content changes on 7 days loading group were statistically significant compared with other groups(P<0.05). (4)Safranin O and Alcilan staining showed that there was a tendency of cartilage differentiation in the experimental group, and the shape gradually increased with time, which was more obvious than that in the control group; The PCM, Col-II and GAG in the experimental group increased gradually with the increase of mechanical stimulation days, which were more obvious than those in the control group. Under conditions of two-dimensional culture, in the early differentiation of mesenchymal stem cells into cartilage, cyclic equiaxial tensile strain can promote the proliferation of BMSCs and the differentiation into chondrocytes. Moreover, cyclic equiaxial tensile strain may promote chondrogenic differentiation through inhibiting the Rho/ROCK 1 signaling pathway.