Study On PAMAM（-NH₂）-BVLD/TPS To Improve The Biocompatibility Of Titanium Surface And Mediate The Formation Of In Vivo Microenvironment

At present,stent implantation is the main ways of clinical treatment of atherosclerosis,however,after implantation,restenosis,thrombosis,inflammation and other problems may lead to treatment failure.These stent-induced clinical complications can be inhibited or resolved by constructing a multifunctional surface microenvironment with anticoagulation,endothelial promotion,and intima repair or regeneration functions on the surface of scaffolds.Studies have shown that the surface properties of materials are important determinants of cell behavior.At present,the regulation of surface microenvironment is mainly focused on the construction of microenvironment in vitro,and the use of protein components in vivo to influence cell behavior is rarely considered.In this paper,the Polyamidoamine(PAMAM)dendrimers was fixed on the titanium surface after alkali activation.Through the amino group of PAMAM,BVLD and TPS were introduced to construct the mediating layer with anticoagulant/endothelial promoting ability,which was used for the formation of in vivo microenvironment.Then,the formation of surface microenvironment was investigated by semi-in vivo experiments.A layer of acellular adsorbent was found on the surface of the mediating layer.In order to explain this phenomenon,the interaction between the mediating layer and proteins was studied.Firstly,PAMAM was connected to the alkali-activated titanium surface,and with the help of its terminal amino functional groups,the PAMAM-BVLD/TPS mediated layer was constructed by introducing bivalirudin(BVLD)and TPSLEQRTVYAK(TPS).Through SEM observation,immunofluorescence staining analysis,surface group structure detection and quantitative analysis of surface functional groups,hydrophilicity and hydrophobicity detection,it was proved that functional molecules were successfully grafted in this study,and the surface hydrophilicity of grafted materials was improved.In the evaluation of blood compatibility,platelet adhesion and activation,activated partial thrombin time(APTT),dynamic coagulation time,hemolysis test results showed that compared with Ti,the number of platelet adhesion in the mediated layer group was smaller,the activation degree was significantly reduced,and the APTT time was significantly prolonged.The results showed that the mediating layer had excellent antithrombotic performance and could effectively inhibit the activity of thrombin and play anticoagulant function.The hemolysis rate of each group was within the safety range(5%)stipulated by the state.In the evaluation of cytocompatibility,the adhesion and proliferation of endothelial cells(EC)and smooth muscle cells(SMC)showed that the number of EC adhesion and proliferation activity on the mediated layer were increased,and the growth of endothelial cells was the best when TPS concentration was 3 M and 5 M.SMC culture showed that the mediating layer did not significantly promote and inhibit smooth muscle proliferation.The results showed that the mediating layer constructed in this study had excellent antithrombotic performance compared with the blank sample.The 180 min semi-in vivo circulation experiment showed that there was a layer of cells adherent to the surface of the mediated layer by the chemotactic ability of recombinant human granulocyte-stimulating factor(G-CSF),but the type of cells needed to be further identified.In addition,there is a layer of non-cellular adsorbent on the surface of the mediating layer,which is preliminarily identified as the protein adsorbent layer.Further,the effects and adsorption of plasma proteins on the above mediating layers with anticoagulant and endothelial promoting functions were studied to evaluate the possibility of the mediating layers forming in vivo microenvironment after implantation.In exploring the influence of the material surface on different proteins in the plasma environment,SEM shows that the adsorption of protein on the surface of the mediating layer reduces the roughness of the material surface.The results of water contact Angle and Micro BCA protein quantitative experiment showed that the Fg content adsorbed on The surface of Ti-OHP was much higher than that on the mediating layer of Ti-OHPBT,which was beneficial to platelet adhesion and activation.The results of ATR-FTIR showed that the protein conformation adsorbed on the sample surface was affected by the concentration of TPS,and the increase of TPS concentration led to the dishelicity of albumin and the change of fibrin conformation,The surface is not conducive to fibrinogen adhesion.In conclusion,the mediating layer with anticoagulant/endothelial promoting function was successfully constructed in this study.In vivo experiments show that it has excellent antithrombotic properties.The interaction between the mediating layer and protein and the adsorption experiment showed that the mediating layer surface was not conducive to the adhesion of fibrinogen,and the fibrinogen content adsorbed on the mediating layer surface was significantly lower than that of the pair.This paper provides a research idea for the formation of cardiovascular material surface microenvironment in vivo.