The Study on the Regulation of Adhesion Protein Adsorption by Material Surface Chemistries through Molecular Dynamics Simulations

Author:Li Tian Jie

Supervisor:wang ying jun


Degree Year:2019





With the developing of regenerative medicine field,the design of biomaterials has entered a new era.Nowadays,studies on new bioadaptable biomaterials with appropriate response reactions has become the focus of tissue repair and regeneration.Particularly,the surface plays a fundamental role in the biological response of materials by regulating protein adsorption and subsequent cell behaviors,which is crucial to the design of novel biomaterials.However,limited by surface complexity and limitations of experimental methods,it is difficult to obtain detailed information in complex processes such as protein adsorption orientation and conformation at the molecular or atomic scale.Focusing on the key scientific issues of chemical biocompatibility on the surface of materials,this thesis studies the molecular mechanism of the regulation for the adsorption of adhesion proteins by the functional groups on material surfaces.Combining basic experiments with all-atom molecular dynamics(MD)simulations,the variation in protein adsorption orientation and conformation were explored at the atomic level.Further analysis of the interaplay between functional groups and integrins on cell surface and the competitive adsorption between proteins revealed the regulation mechanism of key domains within the very protein or different proteins.It is expected to lay a theoretical fundamental for the design of advanced biomaterial surfaces.1)Constructing MC+MD simulation system for protein adsorption on biomaterial surfaces.A molecular simulation system suitable for protein adsorption is necessary for theoretical analysis.In this paper,SAMs with different terminal functional groups(-COOH,-NH2,-CH3 and-OH)were firstly modeled and examined by first-principle calculations and experiments.Furthermore,BSA was applied as the model protein with its orientation initialized by the Monte Carlo(MC)method to build the MC+MD simulation system successfully.The searching time required for the global stable conditions was reduced,compared to the traditional MD method.The reliability of results was highly improved.2)The conformational evolution of adhesion proteins and the cell adhesion activity regulated by material surface chemistry.In order to reveal the regulation mechanism of adhesion protein adsorption on cell behavior,and also to verify the applicability of MC+MD simulation system in the study of protein adsorption,basic experiments were first used to characterize SAMs with different charge and hydrophilicity and the adsorption properties and kinetics of vitronectin(Vn).Then,the cytological response of hMSCs,as model cells,was evaluated on Vn matrix.The MC+MD simulation system was constructed by simulating the functional domain SMB fragment related to cell adhesion in Vn.The results showed that the cell adhesion ability of Vn depended on the different adsorption amount and RGD orientation caused by the conformational changes.The high flexibility of RGD on the charged surfaces led to a larger adhesion area but delayed osteogenic differentiation for hMSCs.Although Vn was unfolded on both negative and hydrophobic surfaces to achieve a higher adsorption density,but the former formed an upright shape which is conducive to cell adhesion,while the latter formed flat multilayers to inhibit cell adhesion.Moreover,the consistency of experimental and simulation results confirmed the applicability of the MC+MD simulation system.3)Regulation for cell adhesion on the adsorbed protein by internal protein domains.The internal structure of the protein is important to its adsorption behavior.In this paper,MC+MD simulation system was constructed using multi-domain fibronectin(Fn)fragments(Fn-III10,Fn-III9-10 and Fn-III7-10),and its adsorption behavior was simulated to further investigate the interaction between the internal domains at the interface with materials.The results showed that the number of Fn-III domains was positively correlated with the stability of adsorption state and conformation.The adjustment of adsorption orientation of Fn fragments by Fn-III depended on the material surface properties.On negatively charged surface,the spatial isolation of Fn-III9 and the polar deflection of Fn-III7-8 increase the activity of RGD sites.On the positively charged and hydrophobic surface,Fn-III9 increased and decreased the activity of both cell-binding sites respectively;On the hydrophilic surface,the binding force was too weak to stabilize the adsorption,which weakened the regulation of Fn-III as well as the cell adhesion activity.4)Biomaterial surface-protein interactions and protein-protein interactions in competitive adsorption between adhesion proteins.In clinical uses,there are types of proteins in the microenvironment on biomaterials.The competition between adhesion proteins is principle for cell-binding functions.Based on the acquired stable adsorption state of Fn-III10and SMB,the MC+MD simulation system of double proteins was constructed by introducing interaction protein.The competitive adsorption behavior was simulated afterward.The biomaterial surface-protein interactions and protein-protein interactions in competitive adsorption between adhesion proteins were further investigated.We have found that the electrically neutral Fn-III10 tends to bind to the negatively or neutrally charged surfaces and to exchange the pre-adsorbed SMB.Fn-III10 and SMB were mainly driven by electrostatic interaction and bound by polar interactions.The basic residues Arg and Lys were important components for guiding and stabilizing.The interactions between proteins regulated the orientation and conformation of pre-adsorbed protein.Especially,the cell adhesion ability of SMB was significantly increased and decreased respectivgely under Fn-III10 adjustment on positively charged and hydrophobic surfaces.To solve the basic problems on protein adsorption and cell response of biomaterials,a universal molecular simulation evaluation system was constructed.The elaboration for molecular mechanisms of the adsorption behavior of adhesion proteins on the surface/interface of materials and protein interaction interface were realized ultimately.These findings are promising to provide systematic theoretical guidance for the surface design of advanced bioadaptable materials.