| Objective:Titanium and titanium based alloys are widely used as various orthopedic implant materials due to their excellent corrosion resistance,high mechanical strength and cell compatibility.In addition,these materials have superior fatigue strength,high wear resistance,and Young's modulus similar to natural human bone.However,implant-associated infections and aseptic loosening after orthopedic surgery are two major challenges after orthopedic surgery.The pathogenesis of implant-associated infections after orthopedic surgery is the adhesion of bacteria to the surface of plants,proliferation and subsequent biofilm formation.The biofilm protects the bacteria from the immune system of host and antibiotic treatment,resulting in implant-associated infections.Failure to combine the prosthesis with the bone tissue can result in aseptic loosening.However,the current implant material commonly used in orthopedics-titanium lacks antibacterial ability and osteointegration ability.Therefore,there is an urgent need to design a metal implant that can simultaneously prevent implant-associated infections and also promote osseointegration to prevent postoperative aseptic loosening.The main strategy currently used to prevent implant-associated infections is to modifythe surface of the titanium by ion implantation,plasma spraying,anodizing and antibiotic coating or antimicrobial polymer.Among them,compared with traditional antibiotics inorganic antibacterial silver has a broad antibacterial spectrum,is less likely to form resistant strains,has low cytotoxicity and satisfactory stability under physiological conditions.Therefore,it has been applied to medical products and instruments in some clinical situations.In the method of imparting antibacterial property to metallic titanium,in addition to modifying the surface of titanium metal with antibacterial metal silver ions or nano silver particles,another important strategy for reducing the occurrence of bacterial infection is to incorporate silver into titanium metal.There are many surface modification methods for enhancing the osseointegration ability of titanium.Among them,acid etching and alkali treatment is a low-cost,effective and highly reproducible surface modification method.After acid etching and alkali treatment,the surface of the material will form unique micro,sub-micron and nano composite structures.In the present study,titanium-silver alloys were prepared by a spark plasma sinteringmethod or a casting method,respectively,and then the surface was subjected to acid etching or/and alkali treatment for surface modification.The surface characterization,antibacterial properties,biocompatibility and osteogenic properties of the two surface modified titanium-silver alloys were evaluated.Methods:Two kinds of titanium-silver alloys were prepared by a spark plasma sintering method and a casting method,respectively.The former was subjected to a mixed etching treatment of hydrofluoric acid and nitric acid?Ti-Ag?AE??,and the latter was subjected to hydrochloric acid etching and sodium hydroxide alkali treatment?Ti-Ag?CT??.Thereafter,the system was evaluated for surface characteristics,ion release,antibacterial properties,biocompatibility,and osteogenic properties of each sample.The surface morphology of the sample was observed by field emission scanning electron microscopy;the surface chemical composition was analyzed by energy dispersive X-ray spectroscopy and X-ray diffraction;the surface roughness was measured by confocal laser scanning microscopy;the hydrophilicity of the sample surface was evaluated by the contact angle measurement;Silver ion release assay,electrochemical analysis and pH determination were used to study the antibacterial mechanism of the samples;the antibacterial ability of the sample was detected by the plate coating counting method after 1 day and 30 days respectively;bacterial activity and degree of bacterial adhesion were observed by the fluorescence of the bacteria and scanning electron microscopy after 1 day of co-culture;cell adhesion was detected by phalloidin and DAPI staining;cell viability and cell proliferation were analyzed by MTT or CCK-8 method;alkaline phosphatase activity and the expression level of the relevant osteogenesis gene and the level of extracellular matrix mineralization were used to evaluate the bone-promoting ability of the samples;by constructing a model of osteoporosis infection associated with the inner plant of the femur of rat,X-ray photographing and internal plant removal of the rolling on plate were used to evaluate the anti-infective effects of samples in the animal.Results:The titanium-silver alloy prepared by the spark plasma sintering method formed micropits and particles with a high silver content on the surface of the Ti-Ag?AE?sample after the acid etching treatment,and t the particles on the surfaces of the Ti-Ag?AE?samples contained a significantly higher Ag content than the substrate and consisted of Ti2Ag and a Ti-Ag inter-metallic phase.The roughness of the titanium-silver sintered alloy after acid etching was significantly increased?p<0.01?,and the hydrophilicity of the titanium-silver sintered alloy treated by acid etching was significantly higher than that of the sample without acid etching?p<0.05?except Ti-5Ag?AE?.In terms of silver ion release,the Ti-Ag?AE?sample initially released a large amount of silver ions,and then the release amount was gradually reduced,and the release amount was higher than that of the titanium-silver sintered alloy without acid etching.The results of electrochemical experiments showed that the Ti-Ag?AE?sample had better corrosion resistance than the cp-Ti and s-Ti?AE?samples,but the corrosion resistance was worse than that of the titanium-silver sintered alloy sample.In the in vitro antibacterial tests,compared to the titanium-silver sintered alloy sample without acid etching,the acid-etched titanium-silver sintered alloy samples on day 1 or were immersed in phosphate buffered saline for 30 days both had significantly better antibacterial rates?p<0.01?,and the antibacterial rates of Ti-3Ag?AE?and Ti-5Ag?AE?remained at 96.53%and 99.04%,respectively,even after 30 days of soaking.Fluorescence staining of live and dead bacteria and scanning electron microscopy confirmed that Ti-3Ag?AE?and Ti-5Ag?AE?samples could not only effectively kill adherent bacteria,but also prevent bacterial adhesion.In the in vitro biocompatibility experiments,cell viability and proliferation of MC3T3-E1 cells showed that the Ti-Ag?AE?samples were not cytotoxic,but did not have the ability to promote cell proliferation?p>0.05?.It was found by field emission scanning electron microscopy that after the cells were co-cultured with the samples for one day,the acid-etched samples showed more pseudopods,and the morphological differences between the samples were not obvious after three days of co-culture.There was no statistically significant difference in alkaline phosphatase activity between cp-Ti and Ti-Ag?AE??p>0.05?,indicating that Ti-Ag?AE?had no effect on cell differentiation.After the acid-etching and alkali treatment?collectively chemical treatment?of the titanium-silver alloys prepared by the casting method,the surfaces of the Ti-Ag?CT?samples exhibited a micro-scale,sub-micron and nano-scale hybrid composite structure and micron-scale and submicron particles.It was found by energy dispersive X-ray spectroscopy that the alkali treatment resulted in the formation of particles with a high silver content on the surface after the acid etching treatment of the Ti-Ag sample.X-ray diffraction detected the presence of?-Ti and TiH2 on the chemically treated samples.The roughness and hydrophilicity of the Ti-Ag?CT?samples were significantly higher than those of the cp-Ti samples?p<0.01?.The silver ion release amounts of Ti-1Ag?CT?,Ti-3Ag?CT?and Ti-5Ag?CT?samples on the first day were 21.20±4.92,45.37±4.56 and145.17±11.70ppb,respectively.The titanium-silver samples released a relatively large amount of silver ions on the first day,and then the amount of release gradually decreased,reaching stability after 7 days.And the pH of the first day was in the range of 6-7.In the in vitro antibacterial tests,Ti-3Ag?CT?and Ti-5Ag?CT?samples showed significantly better antimicrobial activity both on the first day and after immersion in phosphate buffer saline for 30 days?p<0.01?.And the antibacterial rate of Ti-3Ag?AE?and Ti-5Ag?CT?remained above 90%even after 30 days of soaking.Fluorescence staining of live and dead bacteria and scanning electron microscopy confirmed that Ti-3Ag?CT?and Ti-5Ag?CT?samples could not only effectively kill adherent bacteria,but also prevent bacterial adhesion.In the in vitro cell experiments,Ti-Ag?CT?samples showed more cells adhering than cp-Ti samples and exhibited more cytoplasmic extension,more filopodia and flaky pseudopods.However,there was no statistical difference between cp-Ti and Ti-Ag?CT?samples in terms of cell proliferation.However,it was confirmed by calculation of the relative growth rate that the Ti-Ag?CT?samples were not cytotoxic.In the experiments about cell osteogenesis,the alkaline phosphatase activities of Ti?CT?,Ti-1Ag?CT?and Ti-3Ag?CT?samples were higher than those of cp-Ti samples after 7 days of osteogenic induction?p<0.05?.In terms of osteogenesis-related gene expression,Ti-Ag?CT?could promote the expression of alkaline phosphatase,collagen type 1 and osteocalcin gene.In addition,Ti-Ag?CT?could also promote extracellular matrix mineralization.In the in vivo experiments,X-ray images at 7 and 42 days postoperatively showed signs of plant infection in the cp-Ti group,whereas there was no sign of infection in the Ti-3Ag?CT?group.Moreover,in the cp-Ti group,a large number of colonies were formed in rolling plate culture after plant removal,while in the Ti-3Ag?CT?group,no colony was formed in rolling plate culture after plant removal.Conclusion:Titanium-silver sintered alloy sample was prepared by a spark plasma sintering system.The titanium silver sintered alloy sample before acid etching had a limited antibacterial rate and was unsatisfactory.After the acid etching treatment,micropits and particles with a high silver content were formed on the surface of the Ti-Ag?AE?samples,and Ti-3Ag?AE?and Ti-5Ag?AE?samples showed excellent antibacterial properties.Even after immersion for 30 days,Ti-3Ag?AE?and Ti-5Ag?AE?samples showed an antibacterial rate higher than 96%,indicating that they had long-term effective antibacterial properties.In addition,Ti-3Ag?AE?and Ti-5Ag?AE?samples also showed good biocompatibility.A titanium silver casting alloy sample was prepared by a casting method.After chemical treatment,the surfaces of the titanium-silver cast alloys formed micron and submicron mixed pores,spongy nanostructures and silver-containing particles.Ti-1Ag?CT?,Ti-3Ag?CT?and Ti-5Ag?CT?samples showed strong antibacterial properties after 24 hours due to silver ion release and silver-containing particles.Ti-3Ag?CT?and Ti-5Ag?CT?samples remained antibacterial after 30 days of immersion,mainly due to silver-containing particles.In addition,Ti-1Ag?CT?and Ti-3Ag?CT?samples showed a promoting effect on cell adhesion and osteogenic differentiation due to their special surface morphologies.Ti-3Ag?CT?could also play an anti-infective role in animals. |
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