Study On Adsorption Of Typical Serum Proteins On Biomaterials And Its Effect On Adhesion Behaviors Of Cells/bacteria

When biomaterials are implanted into the human body,biomaterial-associated infections and difficulties in tissue integration are two major barriers for their clinical application.However,it should be noted that serum protein adsorption is the initial step prior to following cell/bacterial adhesion to surfaces,thus mediating cell/bacterium-material interactions.Understanding the interfacial interactions among proteins,cells,and bacteria in the microenvironment at the molecular level is essential for biomaterial surface modification.The biological functions of proteins are regulated by their structures,therefore,the mediation mechanism largely depends on the conformation of the adsorbed proteins.Based on the interactions between various biomaterial surfaces and cells/bacteria,this dissertation investigates the mediation effects of adsorbed protein on following cell/bacterial behaviors.Firstly,this project studied the adsorption-associated conformational changes of vitronectin and their effects on cell adhesion behaviors.In order to visualize the adsorbed proteins on biomaterial surfaces,single particle electron microscopy was employed to image and reconstruct protein conformations.It was found that surface functional groups trigger different conformations of vitronectin.On-COOH grafted surfaces,vitronectin showed globular aggregation,exposing a large number of cell-binding domains,and promoted osteoblast adhesion.Whereas on–NH₂ modified surfaces,vitronectin favored to form dimmer,enwrapping some cell binding domains,and exhibited limited promotion for osteoblast adhesion.Furthermore,the integrin related gene expression of osteoblasts was also enhanced by the vitronectin adsorbed on–COOH grafted surfaces,confirming the pronounced effects of protein conformation on cell behaviors.The effect of protein adsorption conformation on bacterial infection was also investigated.Immunoglobulin G?IgG?was selected as a model protein.Results show that IgG adsorption is accomplished by the synergistic effect of electrostatic forces and van der Waals forces.The adsorption orientation of IgG was largely determined by surface charge,and the secondary structures were mainly affected by hydrophobicity of surfaces.Additionally,the exposure levels of cell/bacterial binding domains were related to the protein adsorption orientation.On positively charged surfaces,IgG showed vertical,mainly end-on orientation,and greatly enhanced S.epidermidis adhesion;whereas on negatively charged surfaces,IgG preferred head-on orientation and enhanced macrophage adhesion and phagocytosis.Furthermore,to explore the influence of key serum protein adsorption on bacterial biofilm formation and immune responses,this dissertation investigated and compared the adsorption conformations of two typical serum proteins on newly emerged diamond-like carbon?DLC?films and conventional Ti surfaces,and studied their effects on S.epidermidis infection and early stage immune responses of macrophages.Results show that despite the similar surface physicochemical characteristics of Ti and DLC films,the proteins adsorbed with different conformations.Pre-coating surfaces with proteins dramatically altered surface physicochemical properties and thereby mediated bacterial adhesion.S.epidermidis adhesion was dominated by non-specific interactions and in this case,the electrostatic interactions between adsorbed proteins and bacteria.Furthermore,macrophage adhesion and phagocytosis were largely regulated by the types of the adsorbed proteins.This dissertation also investigated the transformation and mediation roles of key serum proteins during the bactericidal process of Ag⁺,a bactericidal agent with a broad inhibitory spectrum.Results show that the interaction between Ag⁺and IgG decreased the number of adherent bacteria and inhibited bacterial biofilm formation.The inhibitory effect was mainly attributed to the prevention of Ag⁺aggregation.In the meantime,Ag⁺could combine with the Fab domain of IgG,resulting in the unfolding of the protein structure and alteration of the secondary structure,therefore preventing the specific interaction between bacteria and IgG.The results proved the important transformation and mediation role of serum proteins during the bactericidal process of Ag⁺.This dissertation aims to disclose conformational changes of the adsorbed proteins triggered by surface characteristics and analyze the mediation effects of these proteins on subsequent cellular and bacterial behaviors,in order to provide experimental data for future biomaterial surface design.