Construction Of Lactobacillus Functionalized Biofilm On The Surface Of Titanium Implant

With the continuous update and iteration of medical and technology,more and more biological materials are being developed and applied to human disease treatment and health recovery.During the surgical operation,due to the failure of sterilization of the instruments and other reasons,bacterial infection caused the failure of the operation,which would seriously threaten the health of the patient.Conventional treatment methods are injections or taking a lot of antibiotics,but this will not only produce strong side effects,but also cause the production of drug-resistant bacteria,which increases the risk and difficulty of treatment.A large number of new multi-drug resistant bacteria have emerged around the world and are threatening human health,causing large-scale infections and deaths.Although the medical devices and medical materials have been repeatedly disinfected and sterilized,there is no guarantee that the implant site will not be infected by bacteria,which will cause serious complications.Bacteria adsorbed on the surface of medical devices and implants will rapidly multiply and generate a biofilm of bacteria,which will not only destroy the biomedical properties of the implant surface,but also cause lesions in the surrounding tissues.The good surface biocompatibility allows titanium implants to be widely used in orthopedic materials,but the osseointegration ability has always to be improved,and the biological inertia on the surface of the material also seriously affects the success rate of bone implant surgery.There is a great demand for orthopedic implants to prevent infections related to multi-drug resistant bacteria and improve osseointegrity at the same time.Here,we have developed food-grade probiotic-modified implants that can prevent methicillin-resistant Staphylococcus aureus(MRSA)infections and accelerate bone integration.Lactobacillus casei is cultured on the surface of an alkali-heat-treated titanium(Ti)substrate and inactivated by ultraviolet radiation,thereby avoiding sepsis caused by live bacteria.Due to the production of lactic acid and bacteriocins,this inactivated Lactobacillus casei biofilm shows an excellent antibacterial effect of 99.98% against MRSA.In addition,the polysaccharides in the biofilm of Lactobacillus casei stimulate macrophages to secrete a large number of osteoblastic factors,such as Oncostatin M(OSM),and improve the osseointegration of Ti implants.Modification of inactivated probiotics can be a promising strategy for endowing implants with excellent self-antibacterial activity and osseointegration capabilities.The details of the study include the following points:1.Construct a functionalized probiotic biofilm with biocompatibility based on titanium implantsFirst,polish the surface of the titanium sheet and clean it in deionized water;in a sodium hydroxide(Na OH)solution with a concentration of 4 M,react for 1.5 h at a temperature of80 ?,and form a large amount on the surface of the titanium sheet by alkali heating.Empty microstructure;co-cultivation of the original lactic acid bacteria liquid and alkali-heated titanium sheets to form a dense biofilm of lactic acid bacteria bacteria on the surface,and inactivate the bacteria by ultraviolet irradiation;determine the lactic acid bacteria and lactic acid bacteria after 28 days by the immersion method The degree of stability of bacterial biofilm;the antibacterial effect of Lactobacillus casei bacterial biofilm can be observed by the bacterial plate coating method,and the degree of damage of pathogenic bacteria after antibacterial can be observed by observing the morphology of bacteria;detection by the morphology of MTT and cell fluorescence The biocompatibility of the sample itself.2.Functionalized probiotic biofilm with macrophage immune responseCo-cultivate the titanium sheet covered with Lactobacillus casei bacterial biofilm with macrophages for 12 hours;detect the biological toxicity of Lactobacillus casei to macrophages by MTT,and detect the effect of Lactobacillus casei on macrophages by cell fluorescence morphology;use enzymes Linked immunosorbent assay detects the biological immune response of macrophages under the action of Lactobacillus casei bacterial biofilm from the protein level;real-time fluorescent quantitative PCR can detect the role of macrophages in the bacterial biofilm of Lactobacillus casei from the nucleic acid level The internal reaction of the cells generated under the transcriptomics;the intracellular pathway of the immune response of macrophages under the action of the bacterial biofilm of Lactobacillus casei was further determined by transcriptomics;the direction of macrophage differentiation was detected by the loss cytometer.3.Functionalized probiotic biofilm based on titanium implants to promote osteogenic differentiation of mesenchymal stem cellsIn order to determine whether the immune response of macrophages under the action of the Lactobacillus casei bacterial biofilm can promote the osteogenic differentiation of mesenchymal stem cells,the macrophages and the Lactobacillus casei bacterial biofilm were co-cultured for 12 h.Cell supernatant,the cell supernatant is co-cultured with mesenchymal stem cells;the effect of osteogenic differentiation of mesenchymal stem cells on 7 days and 14 days is detected by alkaline phosphatase ALP and Alizarin red staining;real-time fluorescence Quantitative PCR was used to detect the effect of osteogenic differentiation of mesenchymal stem cells from the nucleic acid level;in animal surgery experiments,titanium nails covered with Lactobacillus casei bacterial biofilm were implanted into the tibia of rats,and the bone tissue The pathological section and Mciro-CT model analysis showed that the bacterial biofilm of Lactobacillus casei can promote the osteogenic differentiation of mesenchymal stem cells and accelerate the application of the same type of materials in orthopedic surgery.