Effect Of Cyclic Tensile Strain In Early Differentiation Of BMSCs Into Cartilage Cells In Mice

Objective:To observe the effect of cyclic equiaxial tensile strain in the early differentiation of bone marrow mesenchymal stem cells(BMSCs)into cartilage in mice under conditions of two-dimensional culture,and to investigate the mechanism of cyclic equiaxial tensile strain in early chondrogenic differentiation.Methods:After extraction and isolation of BMSCs from KM mice,identification of cell phenotype and trilineage inducing differentiation were conducted with regular changes of medium and subculture to the 3rd generation.Cells(2 × 105/well)were seeded in a biological plate(BioFlex).According to different treatments,the cells were divided into 2 groups,including group A(non-induction group): cells were cultured with ordinary culture medium for 8 days and treated with mechanical loading for 0,1,3,5 and 7 days respectively,and group B(induction group): cells were cultured with chondrogenic-inducing medium for 8 days and treated with mechanical loading for 0,1,3,5 and 7 days respectively.Experiments were performed using a Flexcell-5000 mechanical loading system(USA),and stretch stimulation was achieved at a sine wave of 0~1%Elong,1Hz and 4h/D,with medium changed at the 2nd,4th,6th and 8th day respectively.At the 8th day after culture,cells in each group were extracted,and the efficiency of cell proliferation in each group was detected by CCK-8.The expression of the Sox9,Col-? and ROCK1 signaling pathway-related molecules was analyzed by RT-PCR.Glycosaminoglycan content in medium changed last was detected using ELISA.Pericellular matrix was observed by safranin O staining and alcian blue staining.Results:1.After primary cells were cultured for 24 hours,partial cells grew adherent to the wall,presenting round-like or polygon-like.After passage,cells grew rapidly,presenting spindle-like,with cell colonies formed in vortex-like growth.Flow cytometry showed that the expression rate of CD45 and Sca-1 on cell surface was less than 3%,and the expression rate of CD11 b and CD44 larger than 95%,suggesting that the isolated cells were almost mesenchyma stem cells with good homogeneity.After trilineage inducing differentiation,the cells were stained,which confirmed that cells could differentiate into osteocytes,chondrocytes and fat cells.2.Detection of cell proliferation rate: With the increase of loading time,proliferation rate in both group A and B increased gradually.At the 0 day after loading,proliferation rate in the group B reduced significantly than that in the group A.With the increase of loading time,proliferation rate in the group B closed to and even exceeded that in the group A.3.RT-PCR: After the treatment of related factors for 8 days,the relative mRNA expression of SOX9,Col-? and ROCK1 in the group B increased as compared with the group A.In the group A,SOX9 showed no obvious change;Col-? expression gradually increased with the increase of loading time;ROCK1 expression decreased gradually with the increase of loading time.In the group B,the relative expression of SOX9 and Col-? increased gradually,while the relative expression of ROCK1 decreased gradually with the increase of loading time.4.Glycosaminoglycan content in the medium changed last was detected by ELISA,revealing that glycosaminoglycan content in the group A showed a decreasing trend,with no statistical significance(P > 0.05),while glycosaminoglycan content in the group B increased gradually.In addition,glycosaminoglycan secretion in the group B increased as compared with the group A.5.Safranin O staining and alcian blue staining demonstrated that both group A and B showed a trend of chondrogenic differentiation,with the shape gradually lengthening as the increase of loading time,and the change in the group B more obvious than the group A.PCM,Col-? and GAG contents in the group A and B increased gradually with the increase of mechanical loading duration,but the change in the group B was more apparent than that in the group A.Conclusion: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/ROCK1 signaling pathway.