Regulation of Protein Molecules through Light-induced Surface Charge and Its Biological Effects
Author:Wang Xiao Zhao
Supervisor:cheng zuo weng wen jian
Surface characteristics play key roles in material-cell or material-tissue interactions in different ways such as regulating the protein adsorption states and behaviors.Therefore,establishing surfaces that response to external stimuli and using their specific stimuli-responsive characteristic to precisely control the adsorption,desorption and conformations of pre-adsorbed protein have been considered important to the optimization of biomaterials performances and understanding of interaction between biomaterials and protein/cell.In this dissertation,light-responsive TiO2 nanostructured films,Si wafers with junctions(Si(p/n))and graphene-on-Si with schottky junction(Gr/Si)surface are used to investigate the light-induced regulation on protein adsorption status and cell responses.Moreover,the mechanism of such protein behaviors,as well as cell sheet detachment on light responsive surfaces are investigated and discussed.On the basis of these works,cell sheets and cell-sheet-derived extracellular matrix(ECM)that can be used in tissue engineering are obtained.The main results are as following:(1)Ultraviolet-light responsive TiO2 films and their cellular responsesA phase-separation-induced self-assembly process was adopted to prepare a carbon quantum dots(CQDs)sensitized TiO2 films.CQDs sensitization could effectively improve the light responses of TiO2 film,through impeding charge recombination and enhancing surface charge accumulation.Such changes at the interface could subsequently result in conformational changes of BSA pre-adsorbed,and eventually promote cell sheet release.After cells reaching confluence,an intact cell sheet could be obtained after 5 min UV365 illumination on CQDs sensitized TiO2 film.That is much faster than that harvested from the pure TiO2 surface(20 min).In addition,the faster release reduces the possible detrimental impact on cell functions during detachment,and helps in maintaining the excellent cellular activity during detachment.(2)Visible-light responsive Si(p/n)surfaces and their cellular responsesThermal diffusion method was adopted to prepare visible-light responsive Si(p/n)surface.The adsorbed protein molecules on Si(p/n)surface,such as bovine serum albumin and collagen-I,show light-induced desorption behaviors.Si(p/n)exhibits obvious photovoltaic effects and results in obvious electrons accumulation on the surface.Molecular dynamic simulation indicates that this variation at the interface could produce enough electrostatic repulsion forces on protein molecules and induce increments of water layer density at the interface.That eventually triggers the release of the adsorbed protein molecules.Such controlled release of adsorbed protein is crucial to cell harvest.It was found that cells grew well on Si(p/n)surface.After 10 min of visible light illumination,nearly 92%single cells were detached from Si(p/n)surface,and an intact cell sheet could be harvested with well-maintained cell viability,cell migration property,osteogenic differentiation and cell surface proteins.This visible light harvested cell sheets method shows promising potential in tissue engineering and regenerative medicines.(3)Visible-light responsive Gr/Si surfaces and their cellular responsesMechanical transferring method was used to fabricate Gr/Si substrate.Gr/Si could absorb visible light and generate photo-carriers,resulting in hole accumulation,i.e.,positively charged surface.When protein molecules were adsorbed on the surface of Gr/Si,their conformations were mainly composed of ordered secondary structures like a-helix and β-sheet;after visible light illumination,the ordered conformations were changed to the disordered,including random coils and β-turn.The mechanism of protein status variation is attributed to the light illumination changes the interaction between Gr and protein molecules.Under visible light,adsorbed protein transferred electrons to Gr,and eventually to Si substrate.This process induced protonation of amino groups,that led to conformational changes of adsorbed proteins.Molecular dynamic simulation demonstrated that light induced conformational changes of BSA and fibronectin could exert detrimental effects on cell adhesion and induce cell detachment.When the light induced protein conformational regulation was used in cell sheet harvest,BMSCs and MC3T3-E1 cell sheets could be rapidly detached from the surface after 10 min visible light illumination.In addition,the harvested cell sheets show good cell viability,well-maintained ECM proteins and cell-to-cell connection,migration property and osteogenic differentiation.Also,easily accessible responsive substrates and well controlled light sources make this approach convenient and safe for cell sheet harvest.This approach not only shows promising potential for cell sheet harvest technology,but also provides new insights into material-protein and material-cell interactions.(4)ECM acquisition and performance based on cell sheetsECM provides a dynamic and complex environment to determine cell fates. Constructing a natural ECM niche is crucial to regulating cell responses.However,it is still a dilemma to recapitulate such complex milieu and the functions of in vivo ECM.On the basis of well-maintained cell viability and functions and completely preserved ECM proteins,a novel approach for fabricating a natural ECM microenvironment is proposed.Through polydopamine connection,different treated cell sheets were tightly immobilized on Ti substrate to be ECM coatings,and their regulatory effects on BMSCs responses were investigated.The cell sheet-derived ECM coatings show well-maintained topographic and biological features of natural ECM,and significantly improve the responses of BMSCs,including initial cell adhesion,further proliferation and eventual osteogenic differentiation.This work provides new insights into material-cell interaction and shows promising potential in tissue engineering and regenerative medicines.In this thesis,light induced effective regulation of protein adsorption behaviors is achieved by dynamically controlling physical and chemical performance of materials at the interface.Their regulated mechanisms are elucidated,and are subsequently used in cell sheets harvest.The light harvested conditions are optimized,cell viability and functions are demonstrated,and their further applications are explored.These are of great significance for the in-depth understanding of the interaction behavior of material-protein,and provide guidance for the dynamic regulation of cell adhesion behaviors.