| Titanium and its alloys are one of the main implant materials for orthopedic applications due to their superior physical and chemical properties and excellent biocompatibility.Although the risk of infection can be reduced by careful management before,during,and after surgery,infection problems may still occur.Once the infection occurs,it will consume a lot of medical resources,increase the patient’s physical pain and economic burden,even lead to death.With the rising problem of antimicrobial resistance due to antibiotic abuse becomes increasingly serious,antimicrobial peptide(AMP)as a new type of antibiotics attracts wide attention.It has become a hot research topic to modify the implant surface with AMP by physical or chemical methods to endow the implant with antibacterial function.In this desseration,we used c-h LF1-11,a human lactoferrin-derived peptide with its N-terminus modified by cysteine to introduce mercapto groups.The modified AMP was then immobilized on the titanium surface through click reaction,and the effects on the antibacterial activity and biocompatibility were explored.The chemical grafting of c-h LF1-11 on titanium method is as follows.The pure titanium surface was activated by Piranha solution first,and then the c-hLF1-11 was grafted onto the titanium surface by click reaction of Mal(maleimide group)with SH(mercapto group)using APTES(silane coupling agent)and MNH(6-(maleimide)hexanoic acid succinimide ester)as the linkers.The characterization and testing are as follows.The physical and chemical properties of the modified titanium surface were characterized through the surface morphology,water contact angle and XPS;the antibacterial activity was tested by incubation of S.aureus and E.coli bacteria on the sample surface,respectively;the mamlian cell biocompatibility was studied by culturing the mouse bone marrow mesenchymal stem cells on the sample.The results showed that the minimum inhibitory concentrations of the antibacterial peptide c-hLF1-11 on S.aureus and E.coli are in the range of 12.5 ~ 25 μg/ml,which is equivalent to the minimum inhibitory concentration of the unmodified antibacterial peptide hLF1-11.This means that the good antibacterial activity of hLF1-11 is maintained after modification.The antibacterial rates of the titanium surface grafted with c-h LF1-11 to S.aureus and E.coli are only 49.9% and 54.3%,but the bactericidal effect to the bacteria adhereed on the material surface is very good.This is probably due to the short hLF1-11 molecule has limited mobility range to kill the free bacteria far from the material surface.To enhance the antibacterial rate of the AMP grafted on titanium surface,PEG was used as a spacer.The effect of different molecular weight Mal-PEGn-NHS on the antibacterial properties was studied.The results showed that the antibacterial property after using the PEG spacer was improved singificantly.The number of bacteria adhered to the surface decreased with the increase of PEG chain length.The antibacterial rates of the three different PEG grafted surfaces on Staphylococcus aureus(SA)were 93.1%,96.0% and 97.5%,respectively,and the antibacterial rates against E.coli were 90.1%,91.3%,93.6 %,respectively.This indicates that c-hLF1-11 has a good bactericidal effect on Gram-negative and Gram-positive bacteria.The adhesion and proliferation of the mouse bone marrow mesenchymal stem cells cultured on the modified surface are comparable with the unmodified surface,which reveals that the good biocompatibility of titanium is maintained.In summary,immobilization antimicrobial peptide on implant surface by chemical grafting with a long chain spacer will not decrease the antibacterial activity of AMP,which can significantly enhace the bacericial compared with the AMP immobilized directly on the implant surface.At the same time,the bioactivity of the implant will not be affected.Therefore,it is of practical implications that the implant antibacterial performance may be improved by chemical modified with AMP. |
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