Study On Controlled Fabrication Of Three-dimensional Micropatterns And Their Effects On Osteogenic Differentiation

The surface topological structures of materials,especially the micropatterns,had an important influence on the growth,adhesion,migration,differentiation and other behaviors of cells.In order to simulate the three-dimensional microenvironment in vivo,the study of micropatterns acting on stem cells has been extended from two-dimensions to threedimensions.However,some challenges reside in the study of stem cells osteogenic differentiation through three-dimensional micropatterns restriction.For example,how to improve the low hosting cells efficiency of three-dimensional micropatterns.Compared with two-dimensionl micropatterns,the influence of three-dimensional micropatterns on stem cells osteogenic differentiation and its mechanism still had been unclear.The in vivo effects of three-dimensional micropatterns on osteogenesis also needed further study.The above key problems have an important research significance for materials promoting tissue regeneration by three-dimensional micropatterns.Focusing on the above problems,firstly,the thesis studied on how to prompting hosting cells efficiency of three-dimensional micropatterns by the optimization of the preparation process.And then the influence of different three-dimensional micropatterns on osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro was studied,as well as the relevant mechanical regulation signal pathways.Moreover,this work further studied the effect of different three-dimensional micropatterns on osteogenesis and its related molecular mechanism in vivo.The main researches were as follows:1.A novel surface modification method of three-dimensional micropatterns was proposed,which realized the limited growth of BMSCs in three-dimensional micro-patterns,and the a more than 90% hosting cells efficiency;2.The differentiation of BMSCs in three-dimensional micropatterns was studied in vitro.With quantitative and semi-quantitative analysis methods,including ALP staining,real-time PCR,WB,and immunofluorescence staining of osteogenesis-related proteins,and revealed that the triangular and the rectangular three-dimensional micropatterns more effectively promoted the osteogenic differentiation of BMSCs compared with the square and circle groups.Moreover,compared with two-dimensional micropatterns,threedimensional micropatterns promoting capacity on the osteogenic differentiation of BMSCs was significantly improved.3.In vitro results of immunofluorescence,q RT-PCR and WB demonstrated that expressions of endonuclear YAP,vinculin,FAK,integrin β1,integrin α1 and β-catenin,in the rectangular and triangular three-dimensional micropattern groups,were higher than those of the circular and square groups,which further validated the molecular mechanism of triangular and rectangular three-dimensional micropatterns regulating osteogenic differentiation by vinculin and its downstream signal.4.The in vivo effects of different three-dimensional micropatterns on osteogenic differentiation were studied by a rat model of skull defect.Immunohistochemistry and q RTPCR results showed that expressions of osteogenesis related proteins OCN,OPN and COL-1,and genetic transcriptions of FAK,integrin β1,integrin α1 and β-catenin,were significantly higher in rectangle and triangle groups,compared with circle and square micropattern groups.Those results demonstrated that triangular and rectangular threedimensional micropatterns could promote osteogenesis by FAK and Wnt signaling pathways in vivo.In conclusion,three-dimensional micropatterns with high restriction efficient on BMSCs growth in was prepared in this study,and its effect on BMSCs osteogenic differentiation in vitro and osteogenesis in vivo were studied.Furthermore,the molecular mechanism of the triangular and rectangular three-dimensional micro-patterns on promoting BMSCs osteogenic differentiation was explored.This work has thus improved the research system of three-dimensional micropatterns affecting BMSCs osteogenic differentiation,as well as provided a new solution for the development of the next generation of threedimensional micropattern-inducible tissue engineering materials based on surface micronano engineering.