Antibacterial Modification Of Ti Substrates Via SI-ATRP And Its Antibacterial Activities And Cytotoxicity Research

ObjectiveTitanium and its alloys have been widely used in orthopedic and dental implants due to their great physical and chemical properties and excellent biocompatibility,generally termed"osseointegration".However,titanium itself does not have antimicrobial ability.In clinical practice,biomaterial-associated infection(BAI)still remains one of the most common complications especially in dental implants and orthopedic applications.Despite strict sterile manipulation during the operative procedures,the use of antibiotics in perioperative period,postoperation infection still occurred frequently.Statistically,the early failure of dental implants had reached approximately 2%while infection rate in orthopedic applications ranges between 2%and 5%,this failure was associated with infection during the surgical placement and the healing process of the surrounding tissue.Therefore,finding suitable methods that entitle implant surface with antibacterial properties is of crucial importance for preventing peri-implant infections.This article innovatively presented a highly antibacterial and without cytotoxicity polymeric quaternary ammonium compounds(PQAC)on titanium surface by surface-initiated atom transfer radical polymerization(SI-ATRP).MethodsThis article carried out a kind of polymeric quaternary ammonium compound by SI-ATRP.First of all,the bromination reaction with dopamine hydrochloride and 2-Bromo-2-methylpropionyl bromide(BIBB)were used to prepare the initiator 2-Bromo-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-propionamide.The initiator was immobilized on titanium surface and dimethylaminoethyl methacrylate(DMAEMA)was grafted on the titanium surface by SI-ATRP.Then,1-Bromothxadecane triggered the quaternary ammonium reaction.What's more,to verify the effect of dopamine,another coupling agent(aminopropyltriethoxysilane,APTES)was grafted on the surface to immobilize BIBB.Then the polyreaction and quaternization were the same as above.Nuclear magnetic resonance(NMR)of 1H NMR spectrum was used to test the initiator 2-Bromo-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-propionamide.The modified surfaces were characterized using contact angle measurements,X-ray photoelectron spectroscopy.Gram-positive Staphylococcus aureus(A.a)and Gram-negative Escherichia coli(E.coli)were used to the antibacterial test.Titanium were stained with Live/Dead BackLight assay kits and observated with fluorescent microscope(FM).Scanning electron microscope was also used to observe the bacteria attachment.By culturing and counting the colony forming units(CFUs)of the two kinds of bacteria on the PlateAgar,we tested the antibacterial ability of modified titanium.MC3T3-E1 was used to evaluate the cytocompatibility.MC3T3-E1 cells were cultured on the bare titanium and two kinds of modified titanium surfaces.Then CCK-8 was used to measure the OD value to evaluate the cytocompatibility.ResultsNuclear magnetic resonance(NMR)of 1H NMR spectrum indicated that the initiator 2-Bromo-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-propionamide was successfully synthesized.Water contact angle measurements could test the hydrophilic and hydrophobic property of each step.After the synthetic initiator was immobilized on the titanium surface,the contact angle changed from nearly zero to 60°.After DMAEMA was polymerized,contact angle decrease to 46°.After quaternization,contact angle increase to 55°.While about the change of the another PQAC group,the contact angle of titanium surface changed from nearly zero to 380 after amination with APTES.After BIBB was inserted,contact angle increase to 75°.And the contact angle changed to 46 ° and 66°after polymerization and quaternization.Water contact angle measurements,X-ray photoelectron spectroscopy(XPS),suggested purposed polyquaternium was successfully grafted on the surface of titanium.Compared to the bare Ti surface,there was a signal at 70.1 eV assigned to Br 3d in the spectra of the Ti-initiator surface,which confirmed that the initiator was successfully immobilized on the surface.After grafting the PDMAEMA brush on the surface,the intensity of the C 1s and N Is signal increased and the signal of Br 3d disappeared.The atomic ratio of C/N from XPS spectra was 54.15/7.5(the atomic ratio of C/N was 62.67/7.64 on the APTES group),which was consistent with the calculated value 8/1 based on the molecular formula.These results confirmed the successful grafting of PDMAEMA on the Ti-initiator surface.After quaternization,the intensity of C 1s signal further increased and the intensity of the Ti 2p signal further decreased,which indicating the successful quatermization.About the antibacterial test,Ti-PQAC possess great antibacterial ability.At the initial adhesion stage,few bacterial cells were detected on the Ti-PQAC surface for both bacteria.Meanwhile,the number of dead bacteria on modified Ti was much higher than bare Ti,indicating modified Ti possess great sterilization ability.At the 24 h of bacteria-biofilm formation stage,more bacteria were detected on bare Ti surface,indicating that Ti implant was prone to support bacterial proliferation and subsequent infection.However,the number of E.coli and Aa on the Ti-PQAC surfaces did not increase as time went on,which was significantly different from that of the bare Ti.Still,few live bacterial cells could be detected on Ti-PQAC surface,and the amount of dead bacteria increased.Similar results showed in SEM images.At the stage of the initial adhesion,the CFUs of the modified surface was markedly decreased compared to bare Ti surfaces,which is consistent with the fluorescent microscopy results,the number of A.a and E.coli on bare Ti increased with the increasing time,indicating that bare Ti implants were prone to support bacterial cell proliferation.The antibacterial rate of modified titanium was up to 90%at three experiment time points.However,there were hardly differences in the number of CFUs on the Ti-PQAC surface at three different experiment time points,which indicated that modified Ti surface possessed relatively long-term antibacterial activity.Bare Ti was set as blank group,dopamine modified PQAC as experimental group,and APTES modified PQAC as control group.The results of the proliferation of MC3T3 cells over 12 h and 1,2,3,and 5 days on the different surfaces showed the viability of cells on modified Ti displayed no statistical differences to the bare Ti group,suggesting modified Ti surface Ti-dopamine-PQAC scarcely influence the proliferation of MC3T3 cells.While the OD value of positive control group Ti-APTES-PQAC was remarkably lower than the other three groups and its growth curve was relatively gentle which means the growing environment of cell was unsatisfactory.ConclusionsIn this work,a highly efficient antibacterial Ti-PQAC surface was successfully fabricated.Antibacterial assay suggest that modified Ti surface possess excellent antibacterial activity.In vitro cytocompatibility tests,Ti-dopamine-PQAC surface showed statistically higher cell viability than control group Ti-APTES-PQAC.In the present study we created a modified surface with great antibacterial ability as well as cytocompatibility which provide a new method for the implant surface.