Preparation Of Dynamic Covalent Hydrogels With Gradient Stiffness And Their Applications In Regulating Cell Behaviors

Hydrogel with adjustable mechanical strength,variable microporous structure and differences complicate components,has become the best option for simulating extracellular matrix materials.However,static and uniformed hydrogel is difficult to accurately simulate the extracellular matrix of dynamic compositional and functional changes.It is an urgent need to design multi-functional hydrogels that overcome these shortages.Therefore,this study aims to prepare a series of hydrogels with adjustable stiffness in order to better mimic extracellular dynamic environment with real growth state of cells,to further explore the interactions between cells and extracellular matrix,with applications in differential screening of stem cell adhesion,migration and differentiation.Hyaluronic acid and gelatin,two natural polymers with good biocompatibility,were used to construct dynamic covalent hydrogels with gradient stiffness.They were characterized and tested in various physics,chemistry and biology aspects to explore their application potentials for tissue engineering.（1）The double network hydrogels with gradient stiffness based on the natural polymer were prepared by the dynamic covalent bond and complexation with CaCl₂.Gelatin derivatives modified with two small molecules,diethylenetriamine（DTA）and 4,7,10-trioxy-1,13-tridecane diamine（TTDDA）were used to react with oxidized hyaluronic acid to form the first layer of chemical cross-linking network.The second layer network was formed by the complexation of CaCl₂with residual carboxyl groups.By modifying functional groups and adding calcium ions,the stiffness of the hydrogels can be regulated in a wide range（compression modulus of 13-170 kPa）.In addition,the hydrogels showed the advantages of good biocompatibility,good self-healing performance,injectability,quick gelation process,easy production,low cost,expecting to have wide application potentials in the biomedical field.（2）Hydrogel platters with gradient stiffness（compression modulus of 1-30 kPa）were prepared by self-healing to form a screening platform for stem cell adhesion,migration and differentiation.A series of hydrogels with different stiffness were prepared by gelatin derivatives and oxidized hyaluronic acid through Schiff base reaction.The hydrogels can be prepared easily and customized into any shapes based on their excellent instantaneous self-healing properties.In this chapter,we introduced a kind of gradient stiffness platform,integrated as"hydrogel plate".Under the guidance of the different stress of uneven stiffness distribution,the rabbit bone marrow mesenchymal stem cells seeded on the hydrogel could migrate preferentially from the medium stiffness hydrogel in the middle to the surrounding 5hydrogels of different stiffness.In addition,differential adhesion and differentiation of stem cells on gradient stiffness hydrogels was found,including neural differentiation on low stiffness hydrogels（GA1.0,1 kPa）,muscular differentiation on medium stiffness hydrogels（GE1.3,10 kPa）,and osteogenic differentiation on the highest stiffness hydrogels（GE1.9,30kPa）.Moreover,the hydrogels with gradient stiffness have many other advantages:excellent injectability,pH responsiveness,good biocompatibility and biodegradability,interpenetrating pore structure,etc.The hydrogels platform with gradient stiffness established in this research can reveal the influences of extracellular matrix on cell growth,migration and differentiation,and further promote the development of stem cell therapy.