Effects Of Chitosan Mechanical Properties And Cholesterol-g-Chitosan On Osteogenic Differentiation Of Cells

Chitosan as bone tissue repair or filling materials has been widely used in clinical application because of good biocompatibility,biodegradability and biological safety.However,the restricted in clinical applications of chitosan is due to poor mechanical properties,slow degradation rate and slow mineralization rate of chitosan.Therefore,the modification of chitosan has become a hot topic at home and abroad.Cholesterol is an important biological macromolecule in vivo since its thermodynamic affinity for the cell membrane as well as the function of cell signaling,cell adhesion,and cell migration.In this study,the influences of the mechanism of the cholesterol-g-chitosan(CHCS)on the differentiation of human bone marrow mesenchymal stem cells(HMSCs)were further investigated,and the effects of mechanical properties of chitosan materials on cell proliferation and differentiation were also evaluated and the results are satisfactory.Based on the above results,a series of chitosan membrane with different mechanical properties were prepared by means of the biological crosslinking agent genipin.The relationship between the mechanical properties of the membrane and the behavior of the cells has been systematically investigated.The results indicate: the cells proliferation rate increased with the increasing of the mechanical strength of the materials.However,the material mechical strength to a certain extent,the cells proliferation rate decreased,but the osteogenic differentiation ability of cells increased with the increasing of the mechanical strength of the materials.Then,the human bone marrow mesenchymal stem cells(HMSCs)were cultured on cholesterol-g-chitosan(CHCS)membrane to explore the effects of cholesterol-g-chitosan and its mechanical properties on osteogenic differentiation of HMSCs.The results showed that the cell proliferation rate on CHCS was significantly faster than that on CS membrance,and the proliferation of the induced cells was significantly higher than that of the uninduced groups.In addition,the results of ALP activity significantly increased of induced groups.All these results suggested that CHCS was more beneficial to the proliferation and osteogenic differentiation of HMSCs than that of CS.Alizarin red calcium nodules staining showed that the osteogenic induced cells were significantly stained with alizarin red and could be visible by the naked eye,while the uninduced cells can't be seen red.And the staining degree of CHCS group was higher than that of CS group.LSCM observation showed that the cell actin on the CHCS film is more directional arrangement than that on the pure CS film.The results of RT-PCR showed that the expression of RUNX2,OCN,ALP,COL-1 increased with the increase of culture time.The gene expression of CHCS group was significantly higher than that of CS group.Western blot results showed that the expression level of osteogenic markers protein of HMSCs seeded on CHCS film was higher than that in seeded on CS film.Finally,the effect of cholesterol on the expression of wif1 and cdh26 gene in Wnt signaling pathway was investigated by RT-PCR and Western blot.The presence of cholesterol was controlled by cyclodextrin-coated.The results showed that cholesterol could improve expression level of the wif1 and cdh26,thereby activating Wnt signaling pathway and affecting osteogenic differentiation of HMSCs.