Role Of Different Microenvironments And Their Effect Mechanism In Regulating Chondrocyte Cell Line Behaviors On Polymeric Surfaces

The physical and chemical properties of cells in different microenvironment will change dynamically during tissue development,and cells in different differentiation stages react diversely to the dynamic changes of ECM.At present,most existing researches focus on the effects of single microenvironment on cells,but few on the multiple microenvironments.Therefore,this study will investigate the effect of stiffness,hydrophilicity and RGD / HAVDI peptides modification on three cartilage cell lines' behaviors,clarify the primary and secondary sequence of three microenvironments as well as their influence mechanism.In this study,three microenvironments were designed and constructed: stiffness,hydrophilicity and peptide microenvironment.PDMS substrates with different stiffness were obtained by adjusting the ratio of Sylgard colloid monomer to crosslinking agent.Surface initiated atomic radical polymerization(ATRP)technology,alcoholysis reaction and Michael addition technology were used to graft poly(2-hydroxyethyl methacrylate)(PHEMA)layer on PDMS substrates with different stiffness to provide similar hydrophilic surface and RGDSC and Ac-HAVDIGGGC peptide modification surface.Cell lines of m BMSCs(stem cell),ATDC-5(progenitor/immature cell)and C28/I2(mature cell)with different chondrogenic differentiation characteristics were used as target cells to investigate their proliferation,adhesion,migration and differentiation on the substrates with different microenvironments.In this study,hydrophilic poly(2-hydroxyethyl methacrylate)brushes were grafted to the surfaces of polydimethylsiloxane(PDMS)with the elastic modulus of 3.66 MPa,101.65 MPa and 214.97 MPa,decreasing water contact angle from 120.4o to 38.5o.The proliferation of three cell lines was faster on the hydrophilic PDMS than the hydrophobic PDMS,but the stiffness of the hydrophilic or hydrophobic PDMS did not have a significant impact on cell proliferation.The increase of the stiffness enhanced cell migration,the cell spread and the gene expression proportion of extracellular matrix/intercellular adhesion molecules(Integrin + FAK/NCAM + N-Cadherin)for all three cell lines,but the increase of the wettability had small promotion in cells migration,spread and gene expression.Moreover,the cartilage-specific genes expression of SOX9 and COL2 downregulated for all three cell lines with the increasing stiffness,and the improvement of hydrophilicity did not affect the expression trend of cartilage specific genes.Cells with different stemness react diversely to different microenvironments of stiffness and peptide sequence.Stronger stemness cells respond more significantly to peptides,m BMSCs cells proliferate fastest at the same time,the expression of cell adhesion molecules(FAK + Integrin,NCAM + N-Cadherin)and the cartilage-specific genes upregulate more than weaker stemness cells.Stiffness has less effect on cell proliferation,and has certain effects on cell morphology,adhesion factors,and chondrogenic differentiation,but it has less effect than peptides on cells.Optimal proliferation occurs on the surface of the RDGSC sequence.In the same stiffness group,the expression of cell-to-cell adhesion molecule(NCAM + N-Cadherin)is highest on the HAVDI peptides surface.On the surface of the same stiffness,the expression of SOX9,COL2 are highest expression on HAVDI peptide surface,which indicates that peptides have a stronger promotion effect on cartilage differentiation.The above results can further understand the influence of microenvironment on cell behavior and the the primary secondary sequence,and reveal the response specificity of different cells to microenvironment.