Construction Of The Microenvironment-responsive Antimicrobial Titanium Implant And Its Antimicrobial Activity

Thanks to its biocompatibility,good corrosion resistance,excellent mechanical and chemical properties,Titanium(Ti)and its alloys are widely used for permanent implants in contact with bone.However,infection associated with these implants still poses a serious threat because of the lack of antimicrobial activity on titanium surfaces.It is an effective method to prevent bacterial infection on the implants through surface modification with antimicrobial agents.However,excessive exposure or continuous low-concentration release of antimicrobial agents in vivo may cause non-negligible side effects,such as potential toxicity and drug resistance.Thus,precise control of the exposure/release of antimicrobial molecules on the surface of titanium implants spatially and temporally is the key to achieve safe and effective antimicrobial effect.To solve this problem,this thesis has constructed microenvironment-responsive smart antimicrobial surfaces that can respond to the changed microenvironment and selectively kill bacteria.Through the on-demand exposure or release of antimicrobial molecules,the surfaces effectively kill bacteria adhering to the implant surface during or after surgery,and exhibited good biocompatibility with normal tissues and the host body,thus achieving safe and effective antimicrobial properties.The main research contents and conclusions of this thesis are listed as follows:(1)Aiming at the common clinical problem of intraoperative bacterial infection,the thesis was based on the hydrogen bond formation law of temperature-responsive polymer p NIPAM at different temperatures to construct a temperature response antimicrobial surface loaded with antimicrobial peptides(AMP)on titanium implant(Ti-p NIPAM-AMP)through ATRP technology and click reaction.The physical,chemical properties,as well as in vitro and in vivo biological properties of the surface were characterized.The results showed that p NIPAM and AMP can be stably modified on the surface of the implant,and Ti-p NIPAM-AMP can selectively exhibit antimicrobial activity and biocompatibility according to the changed temperature.During the operation(at room temperature of 25°C),the p NIPAM chain formed hydrogen bonds with H₂O and stretched,exposing the AMP molecules on the surface and exhibiting excellent antimicrobial activity.In vitro,it inhibited 94.4%of S.aureus and 95.1%of the E.coli,and effectively prevented infection during in vivo implantation;in the body environment(at body temperature of 37°C),the molecular chain of p NIPAM collapsed due to intra-and intermolecular hydrogen bond,embedded the surface AMP molecules and presented good biocompatibility,promoting the adhesion,proliferation of bone marrow mesenchymal stem cells,and tissue healing.(2)Aiming at the high risk of bacterial infection in the initial stage of implantation and the problem of AMP resistance to enzymatic hydrolysis,the thesis was based on the ionization law of phenol cholesterol in tannic acid(TA)molecules at different p H to construct a p H-responsive antimicrobial titanium implant surface(TA/AMP)30 through layer by layer self-assembly method,and its physical and chemical properties and biological properties in vivo and in vitro were studied.The results showed that layer-by-layer self-assembly method can stabilize the TA/AMP multilayers with 30 layers.The electrostatic interactions made the TA/AMP multilayers stably in a non-infectious environment(p H 7.2).Besides,the physical protection of the multilayers improved the enzymatic resistance of AMP.In an infectious environment(p H6.4),the ionization of the phenolic compounds of the TA molecule was inhibited,which made the charge of TA/AMP multilayers unbalanced and quickly released sufficient AMP to kill 88.5%of S.aureus.The bone defect infection model of New Zealand white rabbits further verified that(TA/AMP)30 effectively prevented bacterial infection at the initial stage of implantation(7days),and its surface antimicrobial rate and antimicrobial rate around tissues were as high as97.7%and 92.0%.(3)Compared with the single-response antimicrobial surfaces,the multi-response antimicrobial surfaces present higher response sensitivity and stronger antimicrobial activity.Thus,the thesis used phage as a template to in situ generate and assemble copper nanoclusters Cu NC-Phage with dual responsiveness of p H and H₂O₂,and used it to construct a dual-responsive titanium implant Ti-NC without environmentally stimulating molecules.The research results showed that Cu NC-Phage can exhibit dual-response antimicrobial activity to H₂O₂ concentration and changed p H in the microenvironment.In the microenvironment of bacterial infection(high H₂O₂ concentration,low p H),Cu NC-Phage generated hydroxyl radicals(·OH)with high bactericidal activity through Fenton-like reaction,inhibiting 97.3%of E.coli within 1 h,which was better than that when it only responded to H₂O₂.Ti-NC can inhibit100%of S.aureus and 100%of E.coli within 3 h and effectively inhibit the biofilm of S.aureus s and E.coli within 36 h.Due to the low catalytic efficiency of Cu NC-Phage in a normal physiological environment(low H₂O₂ concentration,neutral p H),both Cu NC-Phage and Ti-NC were non-cytotoxic to bone marrow mesenchymal stem cells and umbilical vein endothelial cells.