Effects Of Dynamic Stiffness On The Differentiation Behavior Of Mesenchymal Stem Cells In Three-dimensional Microenvironments

Although people have made great progress in the field of tissue engineering and regenerative medicine,the repair of osteochondral tissue defects and the exploration of pathological mechanisms are still a challenge.Biomedical hydrogels can be used as a single repair matrix or combined with pre-seeded cells and biologically active growth factors to provide a three-dimensional(3D)microenvironment for cell adhesion,proliferation,differentiation,or controlled release of drugs.More and more studies have shown that in the dynamic biological process,the stiffening of the extracellular matrix is accompanied by growth and development,wound healing and pathological processes.Mesenchymal stem cells(MSCs)also have dynamic interactions with matrix biomechanics.Two-dimensional(2D)cultured cells respond to short-term(a few hours)and long-term(days and weeks)dynamic stiffening.The culture time before and after stiffening is selectively differentiated.While biomedical hydrogel provides an extracellular 3D microenvironment,its mechanical properties such as stiffness and dynamic stiffening can be optimized by design to simulate the 3D mechanical microenvironment in vivo.The main work of this paper is as follows:In Chapter 2,Gel HPAGMA bifunctional gelatin material that can be cross-linked by enzyme/light is obtained by chemical modification of gelatin.The hydrogel prepared by blending it with Gel HPA can obtain hydrogels of different stiffness by changing the concentration of HRP and H₂O₂,the ratio of materials,or the duration of UV light,and can also achieve sequential stiffness control.This hydrogel system has good biocompatibility and controllable stiffness and stiffening properties,which lays the foundation for the subsequent study of the differentiation behavior of stem cells in the three-dimensional microenvironment.In Chapter 3,encapsulating MSCs in hydrogels with different initial stiffness and dynamic stiffening,explore the effect of 3D dynamic mechanical microenvironment on the osteogenic differentiation of MSCs.The results show that the hydrogel materials and cross-linking methods have good biocompatibility,and MSCs have different proliferation,spreading and differentiation behaviors during the dynamic stiffening culture process with different initial stiffness and time sequence.The cytoskeleton in the soft hydrogel is the most spread,which promotes osteogenic differentiation to a certain extent,but its lower stiffness fails to further achieve better differentiation effects.The high crosslinking density of mediate and stiff hydrogels restricts cell spreading and inhibits the osteogenic differentiation of MSCs.The dynamic stiffening hydrogel shows the best osteogenic differentiation effect.In order to explore the mechanism of the conversion of mechanical signals into biological signals in dynamic sclerosis,we performed YAP/TAZ nuclear localization staining.Studies have found that the soft matrix hydrogel before dynamic stiffening can provide MSCs with space for cell spreading,and the increase in stiffness after stiffening provides mechanical stimulation for cells and promotes the differentiation of cells to osteogenic formation.Later stage sclerosis cells spread more than pre-stage sclerosis,and the expression of osteogenic genes is also higher,indicating that the time point of sclerosis is also a regulatory factor for stem cell differentiation.In Chapter 4,encapsulating MSCs in hydrogels with different initial stiffness and dynamic stiffening,explore the effect of 3D dynamic mechanical microenvironment on the differentiation of MSCs into cartilage.Experiments have proved that the hydrogel materials and cross-linking methods have good biocompatibility.In the mediate and stiff hydrogels,the cartilage differentiation of cells was improved than in the soft ones,but the cartilage differentiation of MSCs in the dynamic stiffening group was more significant.Collagen II,Aggrecan and Sox9was higher in the dynamic sclerosis group,and the related gene expression was also consistent with the staining results.Collagen X in the soft hydrogel is higher,and the gene expression is also higher,indicating that the soft matrix induces the hypertrophy of MSCs.The reason why the dynamic stiffening hydrogel is beneficial to the differentiation of cartilage may be that the matrix is beneficial to nutrient transmission and cell metabolism before the stiffening of the matrix,and promotes cell aggregation.After stiffening,the hydrogel restricts cell spreading and further maintains the non-uniformity of MSCs-like chondrocytes.Spread cell morphology.This project reports a hydrogel system with flexibly adjustable stiffness and dynamic stiffening,which can simulate the mechanical microenvironment of the extracellular matrix in vivo,and expand the construction methods of the three-dimensional dynamic mechanical microenvironment in tissue engineering research.In addition,this study provides a scientific basis for the effect of dynamic mechanics on the differentiation behavior of MSCs bone and cartilage,and provides a theoretical basis for the subsequent construction and further optimization of bone and cartilage tissue engineering scaffolds.