Construction Of Animal Infection Model In Situ For Titanium Implant Modified With Antimicrobial Peptides And Characterization Of Their Biological Property In Vivo

Titanium alloys are widely used as bone implant materials due to their great mechanical properties and excellent biocompatibility.However,it is prone to get bacterial infection during the application,which has become a difficult and important point in modern clinical research.Bacterial infection can cause serious consequences,such as leading to disability or even death.Antibacterial titanium implants modified with antimicrobial peptides are able to prevent this infection effectively.However,it still has many problems in vivo due to that the complex microenvironment in vivo will impact the antibacterial properties of the peptide.It is urgent to build a normal and effective animal model to characterize the antibacterial properties of this implant.According to the site of the bacteria and based on the traditional model,in the present study,we build two infection models in rabbit: the model of the bone infection induced by the S.aureus in bone tissue(Animal Model I),and the model of the bone infection induced by the S.aureus on the implant surface(Animal Model II).The physical signs(white blood cells,weight and temperature of rabbits),tissue pathology and microbial detection of the rabbits were characterized to evaluate the infection model.The results showed that the animals used in both two animal models showed a great increase in body temperature,white blood cells and weight loss.The scattered dead bone and infiltrated of the inflammatory cells in the bone tissue damage area were observed by H&E and Giemsa staining assay.And there were a large number of S.aureus on the surface of implants and around the tissue.It illustrated that the infection models were successfully obtained.For the different animal infection models,three kinds of titanium rod-shaped implants based on antimicrobial peptides were constructed.Based on the self-assembly(SAMs)and azide-alkyne click chemical(CuAAC),we prepared Ti-SAMs-AMP and Ti-CuAAC-AMP implants with continuous antibacterial properties.And based on the atom transfer radical polymerization technique(ATRP)and azide-alkyne click chemical technology(CuAAC),we prepared the Ti-PNIPAM-AMP implants which was sensitive to temperature.The antibacterial activity and cytotoxicity of the implants were characterized by S.aureus and mBMSCs in vitro,respectively.The results showed that both Ti-SAMs-AMP and Ti-CuAAC-AMP implants were able to exhibit excellent antibacterial activity and had no cytotoxicity to mBMSCs in vitro,and Ti-PNIPAM-AMP implant only showed antimicrobial activity at 25 ℃ and had no cytotoxicity.We employed the infection model to evaluate the antimicrobial activity of the above implants in vivo.Animal Model I was used to evaluate the antimicrobial activity,pathological change and cytotoxicity to liver and kidney of rabbits for Ti-SAMs-AMP and Ti-CuAAC-AMP implants,because these implants showed continuous antibacterial properties.The results showed that these implants were able to inhibit the bacterial infection in rabbits,and display neglected cytotoxicity to the liver and kidney of the rabbits.In addition,Animal Model II was used to evaluate the antimicrobial activity,pathological change and cytotoxicity to liver and kidney of rabbits for Ti-PNIPAM-AMP implant.The results showed that the Ti-PNIPAM-AMP was able to inhibit the bacterial infection in open environment and have no toxic and side effects on the liver and kidney of the rabbits.In summary,the two infection models in this study were able to evaluate the antibacterial activity and the cytotoxicity to the liver/kidney of the implant with antimicrobial peptides.The models will provide the basis for the application of these implants in clinic.