Effect Of Mechanical Microenvironment On The Morphology Of Collagen And Cadherin

Proteins are not only the important components of organisms,but also one of the basic substances constituting life.Collagen and cadherin are biological macromolecules belonging to extracellular matrix.because of the complex components of extracellular matrix,this Degree thesis chooses two of them,collagen and cadherin,to study their high-resolution morphology and biomechanical and biological response using Atomic Force Microscopy,aiming to study the relationship of structure-properties-biological function.Collagen,as a structural protein,is widely distributed in human body.Mechanical microenvironment regulates the structure and arrangement of collagen monomers self-assembly as driving force.Moreover,biological macromolecules interaction plays critical role in maintaining the biofunction in the whole system.However,the detailed mechanism is still unclear.This dissertation is devoted to study the structure and morphology of collagen self-assembly in vitro and the important role of hyaluronic acid in this process under the stress-strain gradient field.Bovine type I collagen was used as the research model.The collagen solution was incubated under stress-strain gradient field in a 37~oC incubator.By changing the concentration of collagen solution,mechanical loading strength and the ratio of collagen to hyaluronic acid,the morphology and distribution of collagen were investigated using atomic force microscope(AFM).The results showed that collagen fibers were dependent on position,collagen monomer concentration and mechanical microenvironment.The addition of appropriate amount of hyaluronic acid can increase the width and promotes the orientation of collagen fibers along the stress field in order to resist the effect of external force.Cadherin is a cell surface glycoprotein mediating calcium-dependent cell adhesion and communication.It has been identified as a key factor in cell-cell adhesion.Its structure is mainly divided into intracellular region,transmembrane region and extracellular region.Among them,N-cadherin mainly exists in nerve tissue,and E-cadherin mainly exists in epidermis.Because the monomer arm length of cadherin is only more than ten nanometers,and its morphology changes with the change of calcium concentration,which is closely related to its biological function.This dissertation explores the morphology and calcium-dependent properties of N-cadherin using AFM high-resolution and Transmission Electron Microscopy(TEM).It was further studied the interaction by co-culturing N-cadherin with E-cadherin and the adhesion properties of cells on substrate modified by self-assembled N-cadherin.The results showed that the dimer of N-cadherin self-assembly was in the shape of"V",and its opening angle increases as calcium concentration raises which is altered dynamically.The morphology of N-cadherin and E-cadherin were similar and did not affect each other.Appropriate concentration of N-cadherin can promote cell spreading,adhesion and so on.This study would shed light on expanding the application of collagen-based biomaterials in tissue engineering and the study of cell life activities on cadherin modified substrate.Collagen,as a structural protein,is widely distributed in human body.Mechanical microenvironment regulates plays a key role in driving the structure and arrangement of collagen monomers self-assembly as driving force.but the mechanism is not clear.This thesis is devoted to study the changes of the structure and morphology of collagen self-assembly in vitro and the important role of hyaluronic acid in this process under the stress-strain gradient field.In this paper,bovine type I collagen was used as the research object.The collagen solution was added to the mechanical loader and cultured in a 37~oC incubator.By changing the concentration of collagen solution,mechanical loading strength and the ratio of collagen to hyaluronic acid,the morphology and distribution of collagen were observed by AFM.The results showed that collagen fibers were position-dependent,concentration-dependent and mechanical microenvironment-dependent.The addition of appropriate amount of hyaluronic acid can increase the width and orientation of collagen fibers and promote the arrangement of collagen fibers along the stress field to resist the effect of external force.Cadherin is a cell surface glycoprotein that mediates calcium-dependent cell adhesion and communication.It has been identified as a key factor in cell-cell adhesion.Its structure is mainly divided into intracellular region,transmembrane region and extracellular region.Among them,N-cadherin mainly exists in nerve tissue,and E-cadherin mainly exists in epidermis.Because the monomer arm length of cadherin is only more than ten nanometers,and its morphology will change with the change of calcium concentration,which is closely related to its biological function,this thesis uses AFM high-resolution model to explore the morphology and calcium-dependent changes of N-cadherin,co-culture N-cadherin with E-cadherin and explore their interaction,and quantify the substrate of N-cadherin.The adhesion of cells modified by self-assembled N-cadherin was further studied by high-resolution transmission electron microscopy.The results showed that the dimer of N-cadherin self-assembly was in the shape of"V",and its mouth opening size increased with the increase of calcium concentration and could change dynamically.The morphology of N-cadherin and E-cadherin were similar and did not affect each other.Appropriate concentration of N-cadherin can promote cell spreading,adhesion and so on.The research results of this dissertation can expand the application of collagen-based biomaterials in tissue engineering and it is of great significance for the study of cell life activities on N-cadherin substrate.