The Antibacterial And Antibiofilm Properties Of CoCrMo Alloy Coating With GO And ε-PLL And Its Biocompatibility

BACKGROUND:Artificial joint replacement has superior clinical manifestations and titer ratio,which can enhance the living conditions of patients with end-stage joint disease and alleviate the family and social burden of patients.Therefore,it gained the reputation of “The Operation of the century”.Periprosthetic joint infection(PJI)is one of the most common and dangerous complications after the artificial joint replacement.PJI accounts for 25% to 38% of all complications after Total knee arthroplasty(TKA)alone.PJI is mainly caused by bacteria and biofilms produced by bacteria.The incidence of PJI is still around 1% to 3% despite a series of measures taken by clinicians,including the use of perioperative antibiotics,intraoperative use of antibiotic cement,and improvement of the operating room environment.With the gradual increase of the elderly population,the number of artificial joint replacements is increasing year by year,and so is the number of PJI.Cobalt chromium Molybdenum(CoCrMo)alloy has been one of the main materials for artificial joints for many years.As a cobalt-based alloy,CoCrMo alloy not only has excellent mechanical properties,such as corrosion resistance,fatigue resistance,wear resistance,and tensile strength,it also has good biological properties.At present,CoCrMo alloy is widely used in the manufacture of artificial hip and knee implants,removable dentures,and metal fixed brackets,etc.In recent years,scholars have done a lot of research on surface modification of CoCrMo alloy,but most of these studies focus on improving the biocompatibility of CoCrMo alloy,and there are few studies on antibacterial.With the increasing number of PJI,antibacterial and antibiofilm coatings have become a new research direction in orthopedics and stomatology.Graphene oxide(GO)is the most widely studied Graphene derivatives,and its edge defect sites are rich in oxygen-containing functional groups,such as carboxyl(-COOH),carbonyl(-C=O),and hydroxyl(OH).The antibacterial properties of GO were first discovered in 2010,and the antibacterial properties of GO have been widely studied since then.ε-poly-l-Lysine(ε-PLL)is a natural polymer synthesized by microorganisms and characterized by peptide bonds between the carboxyl group of Lysine and the ε-amino group.ε-PLL is widely used in various food,medicine,and electronic products as an additive.It was found that ε-PLL had a good antibacterial effect on Gram-positive bacteria(GPB)and Gram-negative bacteria(GNB),fungi,mycoplasma,and chlamydia,and ε-PLL had high biosafety.At present,research on ε-PLL is mainly focused on the field of food anti-corrosion,and there are few studies on other fields.Both GO and ε-PLL have antibacterial effects,and GO has a negative charge in the solution while ε-PLL has a positive charge.In this study,GO and ε-PLL composite coatings were prepared on the surface of CoCrMo alloy by the electrodeposition method.Gram-positive Staphylococcus aureus(S.aureus)and Gram-negative Escherichia coli(E.coli),which are common in clinical PJI,were used as research objects to study the antibacterial and biofilm resistance of CoCrMo alloy surface after coating.The effects of GO and ε-PLL coating on the proliferation and differentiation of rat bone mesenchymal stem cells(BMSCs)were studied.It provides new ideas for the clinical prevention of PJI.Part Ⅰ: The preparation and characterization of GO and ε-PLL coatings on CoCrMo alloy【Objective】GO and ε-PLL coatings were constructed on the surface of CoCrMo alloy by the electrodeposition method,and the surface properties of the coatings were analyzed.【Method】GO and ε-PLL coatings were prepared on the surface of CoCrMo alloy by the electrodeposition method.After the coating was prepared,Atomic Force Microscope(AFM),Raman Spectroscopy,Field Emission Scanning Electron Microscopy(FE-SEM),Attenuated Total Reflection Flourier Transformed Infrared Spectroscopy(ATR-FTIR)was used to characterize the coating and verify whether it was prepared successfully.Then the properties of the coatings were analyzed by contact angle measuring instrument,nanoindentation instrument,nano scratch instrument,etc.【Results】After the GO coating is constructed on the surface of CoCrMo alloy,the characteristic cloud-shrouded GO folds can be seen by FE-SEM.After ε-PLL coating,the CoCrMo surface presents a dense film,and the CoCrMo/GO/ε-PLL surface presents a particle aggregation.Raman spectroscopy and ATR-FTIR were used to detect the surface of CoCrMo alloy,and it was confirmed that GO and ε-PLL were successfully coated on the surface of CoCrMo alloy.By AFM and contact angle measuring instrument,it is found that the surface roughness and hydrophilicity of CoCrMo alloy are improved obviously after coating with GO and ε-PLL.Compared with the control group,the elastic modulus and Vickers hardness of CoCrMo alloy coated by GO and ε-PLL did not decrease significantly,and the surface friction of the experimental group was significantly better than that of Bioactive Glass(BG)after coating.It was found by SEM-EDS that CoCrMo alloy was stable after voltage action.【Conclusion】GO and ε-PLL can be successfully coated on the surface of CoCrMo alloy by the electrodeposition method.After coating,CoCrMo alloy has better coating adhesion,elastic modulus,and Vickers hardness,the surface is smoother,the hydrophilicity is better,and the CoCrMo alloy is stable under the action of voltage.Part Ⅱ: Study on antibacterial and antibiofilm properties of GO and ε-PLL coatings on CoCrMo alloy【Objective】To study the antibacterial and antibiofilm properties of GO and ε-PLL coatings on CoCrMo alloy,by using S.aureus and E.coli which are common in PJI.【Method】S.aureus and E.coli were cultured on the surface of each group for 24 and 48 h.The antibacterial effects of GO and ε-PLL on the surface of CoCrMo alloy were verified by FE-SEM observation,plate colony counting,live/dead bacterial staining,Alma Blue staining.FE-SEM,FITC-Con A staining,and Crystal Violet staining were used to observe the formation of biofilm on the surface of each group,and the antibiofilm effect of the coating was analyzed.Then,PCR was used to detect and analyze the effects of GO and ε-PLL on the expression of Nuc,Hlb of S.aureus virulence genes and biofilm-associated gene Dlt B and E.coli virulence genes Omp A and Fli A and biofilm-associated gene Fim H after CoCrMo alloy surface coating.【Results】GO and ε-PLL on the surface of CoCrMo alloy have an obvious antibacterial effect on S.aureus and E.coli.By FE-SEM observation,it was found that the number of bacteria adhered to the surface of the samples in the experimental group was significantly reduced,and a large number of bacteria adhered also died.In the CoCrMo/GO group,S.aureus and E.coli cells were perforated,and content leakage was the most common.S.aureus and E.coli cells atrophy and rupture were most common in CoCrMo/ε-PLL and CoCrMo/GO/ε-PLL groups.Plate colony counting,live/dead bacterial staining and Alma Blue staining showed that the coating in the experimental group had a significant antibacterial effect compared with the control group(P<0.05).Furthermore,by FITC-Con A staining and Crystal Violet staining,it was found that GO and ε-PLL coating on the surface of CoCrMo alloy could significantly reduce the formation of biofilm on the surface of CoCrMo alloy.PCR results showed that GO and ε-PLL could significantly reduce the expression of s.aureus and E.coli virulence genes and biofilm-related genes after CoCrMo alloy surface coating,which proved the antibacterial and antibiofilm properties of GO and ε-PLL after CoCrMo alloy surface coating at the molecular level.【Conclusion】The coating of GO and ε-PLL on CoCrMo alloy has obvious antibacterial effect on S.aureus and E.coli and can reduce the formation of biofilm on the surface of CoCrMo alloy,providing a new idea for clinical treatment and prevention of PJI.Part Ⅲ: Study on the surface biocompatibility of CoCrMo alloy coated with GO and ε-PLL【Objective】To study effects of GO and ε-PLL on the adhesion,proliferation,and osteogenic differentiation of BMSCs in SD rats after CoCrMo alloy coating.【Method】BMSCs of P3～P5 generations were selected as experimental cells.The surface materials of the control group and experimental group were sterilized by Co60,then BMSCs were inoculated on the surface of each group and cultured until the corresponding time.FE-SEM,immunofluorescence staining,CCK-8 cell proliferation detection,live/dead cell staining,alizarin red-S staining,RT-PCR,Western Blot,and other experimental methods were used to detect BMSCs adhesion,proliferation,and osteogenic differentiation on each material surface.【Results】FE-SEM observation showed that BMSCs could adhere to and proliferate on the surface of each group.The growth state,cell morphology,and proliferation of BMSCs in the CoCrMo/GO group were better than those in the control group.Immunofluorescence staining results showed that CoCrMo/GO and CoCrMo/GO/ε-PLL groups that containing GO were significantly superior to CoCrMo alloy and CoCrMo/ε-PLL groups in terms of cell morphology,cell proliferation,and fluorescence intensity of adhesion spots.CCK-8and live-cell count showed that GO could significantly promote the proliferation of BMSCs,while ε-PLL coating had no toxicity to the proliferation of BMSCs.Alizarine redS staining,RT-PCR,and Western Blot analysis showed that CoCrMo/GO group and CoCrMo/GO/ε-PLL coating could promote the osteogenic differentiation of BMSCs and promote the expression of some osteogenic genes.【Conclusion】GO and ε-PLL coating on the surface of CoCrMo alloy are safe and non-toxic for BMSCs adhesion,proliferation,and differentiation,with high biosafety.Among them,CoCrMo/GO group and CoCrMo/GO/ε-PLL that contain GO can promote the adhesion and proliferation of BMSCs on the surface of CoCrMo alloy and can improve the differentiation of BMSCs to osteogenesis,especially at CoCrMo/GO group.