Research Of Construction Of Viscoelastic Cell-based Model And The Influence Of Immune Response Induced By Its Mechanical Properties On Osteogenic Differentiation Of Stem Cells

The viscoelasticity of cell fate and functional state in the immune system plays an important role in the regulation of cell function and the development of many diseases.The change of tissue/ECM viscoelasticity is likely to play a key role in the progression of diseases.Therefore,matrix viscoelasticity has become an important design parameter of biomaterials in the field of regenerative medicine.Through regulating the hydroxypropyl cellulose ester liquid crystal compound structure of rigid/flexible part of the fabric soft material preparation of liquid crystal,this thesis is going to build the LCD substrate model(CnPC)with viscoelastic properties,and with a series of changes as a gradient of modulus of elasticity.It will also stimulate the soft contact and mechanical response of cells in the body mechanics microenvironment,and explore RAW264.7 macrophages induction,and response Phenotype and function of M1/M2 after base force stimulation.Meanwhile,this dissertation is going to analyze the mechanism of immune-mediated osteogenic differentiation of bone marrow mesenchymal stem cells(r BMSCs).The results show that of the 5 groups of CnPC prepared in this research,when the alkyl number n of the flexible side chain of CnPC is 2,4,6,8,all the CnPC exhibits the color texture of cholesteric liquid crystal under polarizing microscope,and its microscopic morphology presents the wrinkled texture and shows the dynamic rheological properties of viscoelasticity.The elastic modulus G’ and the viscous modulus G’’ have strong frequency dependence,and G’’ is higher than G’.When the alkyl number of CnPC side chain n increases to 12,C12 PC shows a dark field under polarizing microscope and still exhibits viscoelastic dynamic rheological properties.However,the values of G’and G’’ change in different frequency ranges.The results of co-culture of RAW264.7 macrophages and CnPC show that RAW264.7 is time-sensitive in response to mechanical stimulation of substrate.After 24-hour co-culture,RAW264.7 can sense and respond to mechanical stimulation of CnPC liquid crystal substrate with different elastic moduli,resulting in changes in cell behavior,including cell morphology,cell migration rate,macrophage gene expression and cytokine secretion.After the cells were co-cultured with the liquid crystal substrate for 1 day,they were grown on RAW264.7 cells of C2PC～C12PC.M2-type macrophages were the predominant cells.The macrophages on the surface of C2 PC,C4PC and C12 PC had fewer pseudopodia,and the cell morphology was mainly spindle-shaped,which was similar to M2-type cells.M1-related genes TNF-A,INOS and IL-Lβ of RAW264.7 planted on five groups of CnPC liquid crystal substrates show high expression levels on day 3,and M1-related genes C6 PC and C8 PC are relatively higher.M2-related genes IL-10,ARG-1 and CD206 are highly expressed on day 1,and the expression levels of M2-related cytokines in C2 PC and C4 PC were higher than those in other groups,while the expression levels of M1 and M2-related genes are down-regulated on day 7.When r BMSCs are co-cultured with CnPC liquid crystal substrate,the high elastic modulus liquid crystal substrate C12 PC can promote the high expression of OST and ALP and the osteogenic differentiation of r BMSCs.When r BMSCs are co-cultured with CnPC liquid crystal substrate in conditioned medium,the osteogenic differentiation ability of r BMSCs planted in 5 groups with different elastic moduli is significantly improved,indicating that the immune microenvironment exerts an important influence on the osteogenic differentiation of r BMSCs.The CnPC liquid crystal substrate with adjustable viscoelastic properties constructed in this thesis can induce different immune microenvironments through the change of elastic modulus,regulate the phenotypic polarization of RAW264.7macrophages,and then have an effect on the osteogenic differentiation function of r BMSCs.The results can provide a reference for clarifying the mechanism of M1/M2 type transformation in macrophages regulated by viscoelastic stimulation,and provide a strategy for cell culture-based immune microenvironment regulation in vitro.