Study On Antibacterial And Osteogenic Properties Of Ti-Cu Alloys With Different Cu Contents After Surface Modification

Objective:Pure titanium is one of the most widely used implant materials due to its low density,high specific strength,excellent corrosion resistance,good biocompatibility,non-magnetic,non-toxic and other excellent properties.However,pure titanium,as a biologically inert material,has no antibacterial properties,which may cause poor local osseointegration and lead to the infection of bacteria.The implant neck and other areas were easily adhered by bacteria and cause peri-implant inflammation.Therefore,it is particularly important to improve the long-term antibacterial activity of implants.Studies have reported that surface modification of implants,such as surface coating with antibiotics,could inhibit bacterial infection after implantation and reduce the occurrence of peri-implant inflammation.However,the limited coating area and the poor binding effect of antibacterial active substances could significantly limit the load capacity,and lead to decomposition and loss,inhibiting the development and application of implant surface coatings.Therefore,preparing the titanium implant containing overall long-term antibacterial properties,improving the biological activity of the pure titanium surface,and improving the ability of early osteogenesis,can effectively reduce the incidence of bacterial infections after implantation and play a great significance on improving patient quality of life.Copper contains the high efficiency,durability and broad-spectrum antibacterial properties,which is also an essential trace element for the human body.Copper can prevent osteoporosis,promote osteogenic differentiation.Our previous research shows that adding 5%copper to the titanium alloy can effectively kill common pathogenic bacteria such as S.aureus and E.coli.Moreover,the anodic oxidation technology is easy to operate,low cost,and easy to control.It has become one of the efficient ways to change the surface morphology of titanium and titanium alloys and enhance their osteogenic properties.Anodizing can not only enhance the wear resistance and corrosion resistance of pure titanium implants,but also form a surface morphology with micro/nano scale structure.Our team has showed that Ti-Cu alloy performed good antibacterial properties,corrosion resistance,excellent biocompatibility and bone formation ability,and has good application prospects as implant materials.Based on these studies,we used anodizing technology to modify the surface of titanium-copper alloys containing three different copper content Ti-x Cu(x=3,5,7 wt.%)alloys.Comprehensive research was performed to analyze and evaluate of material properties,biocompatibility,antibacterial properties and osteogenesis in vitro.The new surface-modified titanium-copper alloy with the best comprehensive properties such as antibacterial properties,bone-forming ability and biocompatibility was screened out.Then we selected Ti and Ti-5Cu alloy implants to implant in the mandibular premolars of beagle dogs for 3 months to observe the effect of the alloy on the bones in vivo.We provided an experimental basis for exploring the clinical application of the optimized copper content Ti-5Cu alloy.Methods:Firstly,materials of titanium-copper alloys with different copper content were prepared with surface modification treatments.Then the physical properties of the materials were evaluated,and the biological properties of the materials were comprehensively evaluated from various aspects such as antibacterial experiments,cell experiments and animal implantation experiment.Preparation of Material:Ti-x Cu(x=3,5,7 wt.%)alloys were produced by arc melting pure titanium and pure copper in a vacuum consumable furnace.The ingot was heat-treated at 740°C and cold-worked into a bar at room temperature.The material in control group was selected with commercial secondary pure titanium(Ti).The material was cut into discs with a diameter of 10 mm and a thickness of 2 mm,and graded mechanically polished to#2000 with silicon carbide sandpaper.Then the titanium-copper alloys were put into 3%hydrofluoric acid solution for anodizing to obtain anodized+HF acid etching Ti-3Cu alloy(Ti-3Cu);anodizing+HF acid etching Ti-5Cu alloy(Ti-5Cu)and anodized+HF acid etching Ti-7Cu alloy(Ti-7Cu).The pure titanium of the control group was subjected to sandblasting with alumina particles and then acid-etched to obtain sand-blasted and acid-etched pure titanium(Ti).Material properties:The surface morphology of the material was observed and analyzed through the laser confocal scanning electron microscope,and the surface roughness parameters of the material was calculated.The evaluation and analysis of the micro/nano surface of the sample and the cross-sectional microstructure of the alloy were carried out.According to the ISO 10993-12 international standard,Ti-x Cu(x=3,5,7 wt.%)alloys were immersed in physiological saline and immersed at 37°C for 1,3,7,14,21,28 days.And then,the inductively coupled plasma mass spectrometer was measured for the content of copper ions in the solution,and the wettability of the material surface was measured by the shape image analysis method.The contact angle through the image of different alloys was measured to compare the hydrophilic and hydrophobic properties of the materials.Antibacterial experiment:S.aureus was used as the experimental strain.Ti was used as the control to evaluate the antibacterial activity of Ti-x Cu(x=3,5,7 wt.%)alloys.The antibacterial rate of the titanium-copper alloy,the influence on the change of bacterial morphology and the formation of bacterial biofilm were studied from three aspects:quantitative antibacterial test,biofilm observation and bacterial morphology inspection.S.aureus was co-cultured with Ti-3Cu,Ti-5Cu,Ti-7Cu and Ti at 37°C for 24 hours,and the viable bacteria attached to the surface of the material and those in the surrounding medium were counted by spreading the agar plate to calculate the antibacterial rates.The viability of bacteria was detected by LIVE/DEAD(?)Bac Light TM Viability Kits L7012and observed by laser confocal scanning microscope.DAPI,SYPRO orange stain and Con A were used to stain the bacterial biofilm in sequence,and then observed with a laser confocal scanning electron microscope and performed by three-dimensional reconstruction analysis.Scanning electron microscope was used to observe the effect of co-incubation with different materials on the morphology of Staphylococcus aureus.The changes of Ti-x Cu(x=3,5,7 wt.%)alloys on the internal microstructure of bacteria were detected by transmission electron microscope,and the antibacterial mechanism of the experimental materials was deeply analyzed.In vitro experiment:The biocompatibility of titanium-copper alloys with different copper content was investigated via MC3T3-E1 cell proliferation,adhesion and apoptosis experiments;The ALP activity and the expression of related osteogenic proteins under co-cultured cells with Ti or Ti-x Cu were detected to determine the promotion of bone performance in different materials.MC3T3-E1 cells were co-cultured with the sterilized Ti-3Cu,Ti-5Cu,Ti-7Cu and Ti.The CCK-8 experiment was used to determine the cell proliferation rate and examine the level of cytotoxicity in material.After co-cultivating Ti-3Cu,Ti-5Cu,Ti-7Cu and Ti with the cells for 4 hours and 24 hours,the MC3T3-E1cells were stained by rhodamine-labeled phalloidin and DAPI,observed by fluorescence microscope to show surfaces of materials and changes in the cytoskeleton structures.The FITC/PI double staining method was used to determine apoptosis of the cells when the cells were incubated with the material for 3 and 7 days,and the early apoptosis rate of the cells was determined.The kit of alkaline phosphatase assay was used to detect the levels of ALP activity on 3 and 7 days.The RT-q PCR experiment was used to determine the m RNA expression levels of Runx2,OPN and Collagen I related to cell osteogenesis on 3and 7 days.In vivo experiment:Under general anesthesia,the first to fourth premolars of the mandible on both sides of the Beagle were extracted.Three months after natural healing,two Ti-5Cu implants were implanted in the unilateral mandible of each dog.In the same part of the contralateral mandible,two Ti implants with the same size were implanted.After that,the animals were observed regularly after implantation.Three months after implantation,the animals were euthanized.And grossly observation,X-ray,Micro-CT detection and Methyl blue-acid fuchsin staining experiments were performed.Results:Material properties:The actual Cu compositions in Ti-3Cu,Ti-5Cu and Ti-7Cu alloys are 3wt.%,4.82wt.%and 6.6wt.%.After surface modification treatment,the surface of Ti-3Cu,Ti-5Cu and Ti-7Cu alloys forms evenly distributed multi-level pit-like cavities.The bottom of the cavity has obvious micropores of different sizes,with diameters ranging from 500 nanometers to 2 microns.Laser confocal scanning electron microscopy combined with three-dimensional surface reconstruction demonstrated that the average roughness of the four groups of materials was about 1.0 micron,and there was no significant difference in roughness between the groups(p>0.05),and they were all moderately rough surfaces.The cross-sectional scanning electron microscope results showed that as the increase of the copper content in the alloy,the Ti₂Cu precipitates in the alloy matrix increased correspondingly,the distance between the Ti₂Cu strips decreased,and the arrangement became tighter.The water contact angle measurement results showed that four groups of material alloys were all greater than 90 degrees,and they were all hydrophobic surfaces,and there was no significant difference between the groups(p>0.05).In the Cu²⁺dissolution test,the Cu²⁺release of Ti-3Cu and Ti-5Cu alloys in the first 3 days increased significantly with the extension of time;from 3 to 28days,the Cu²⁺release of Ti-3Cu increased slowly and Cu²⁺in Ti-5Cu alloy release basically unchanged.The content of Cu²⁺release on the surface of Ti-7Cu was relatively high and gradually increased with time,reaching 475.4mg/L after 28 days,which was much higher than the content of Cu²⁺on the surface of Ti-3Cu and Ti-5Cu.Antibacterial experiment:The colony counting method was used to show the antibacterial activity of Ti-Cu alloy.The surface of Ti-3Cu had an antibacterial rate of92.5%and performed the antibacterial activity.The antibacterial rate of Ti-5Cu and Ti-7Cu surfaces were 99.4%and 99.9%respectively,which had extremely strong bactericidal activity.These results showed that the antibacterial rates of Ti-5Cu and Ti-7Cu were both greater than that of Ti-3Cu,and their antibacterial rates were similar.The results of bacterial viability showed that both Ti-5Cu and Ti-7Cu had the effect of preventing bacterial growth and bacterial biofilm formation.The fluorescent staining of the triple staining system(DAPI+SYPRO+Con A)showed that the continuous bacterial biofilm was formed on Ti surface,which also provided convenient conditions for more bacteria to duplicate.On the contrary,a small number of bacteria were scattered on the surface of Ti-3Cu,and almost no bacteria survived on the surface of Ti-5Cu and Ti-7Cu,no extracellular polysaccharides and proteins were observed.Scanning electron microscopy results showed that a large number of S.aureus adhered to Ti surface,the bacteria were spherical,and a large number of bacteria gathered and accumulated in clusters,arranged in clusters of grapes.The surface of Ti-3Cu,Ti-5Cu and Ti-7Cu all showed adherent broken and dead bacteria,and the bacteria collapsed and shrank obviously.Transmission electron microscopy results showed that after 24 hours of co-cultivation of Staphylococcus aureus with Ti-3Cu,the bacteria shrank and deformed,and the surface of the bacteria was damaged and broken.After Ti-5Cu and Ti-7Cu interacted with S.aureus for 24 hours,the surface of the bacteria ruptured,a large amount of cytoplasm was lost,the inside was hollow,and the bacteria died.The SEM results of the bacteria after 24 hours of incubation with the material were also consistent with the results of the aforementioned antibacterial experiment.In summary,Ti-Cu alloys had certain antibacterial properties,and the overall antibacterial properties were Ti-7Cu?Ti-5Cu>Ti-3Cu.In vitro experiment:The four groups of materials were co-cultured with MC3T3-E1 cells,and CCK-8 experiment showed that cell proliferation was not significantly affected,and Ti-5Cu cell proliferation was the fastest on the 7th day.According to the classification of toxicity evaluation standards,the RGR%of Ti-3Cu and Ti-5Cu were both grade 0,and Ti-7Cu was grade 1.The results of cell adhesion experiments showed that,compared with pure Ti,the surface morphology of Ti-Cu alloys were conducive to the adhesion and extension of MC3T3-E1 cells.The cells were tightly connected,and the extension was good.In the apoptosis experiment,it was found that all three Ti-Cu alloys had good biocompatibility and met the standard requirements of implant materials.Among them,the apoptosis rate of the Ti-5Cu group was the lowest.The detection of proteins related to osteogenic performance found that the Ti-5Cu group and Ti-7Cu group could significantly increase the level of intracellular alkaline phosphatase.At the same time,all Ti-5Cu materials could up-regulate the m RNA expression levels of osteoblast-related genes Runx2,OPN and Collagen I.In vivo experiment:The animal implantation experiment showed that the osseointegration ability of Ti-5Cu implant was similar to that of Ti group under X-ray observation,Micro-CT observation and Methylene blue-acid fuchsin staining.Ti and Ti-5Cu exerted good osseointegration properties,and obvious osteolysis occurred around the implant.Conclusions:1.The surface properties of Ti-x Cu(x=3,5,7 wt.%)alloys and Ti were similar,and the average surface roughness was similar,both of which were moderately rough surfaces.The water contact angles of Ti-x Cu(x=3,5,7 wt.%)alloys and Ti were both greater than 90 degrees,all of them were hydrophobic surface characteristics,and there was no statistical difference between the four groups.2.Following with the increase of copper content in the Ti alloy,the Ti₂Cu precipitation phase in the alloy matrix increased correspondingly,the distance between the Ti₂Cu strips decreased,and the arrangement became tighter.3.About the Cu²⁺release rate of the three materials,Ti-7Cu released the fastest and the most.However,the Cu²⁺ion velocity of Ti-5Cu alloy was stable and the release amount was moderate.Ti-3Cu alloy released the least of the three group.4.Ti-3Cu had bactericidal activity,the surface of Ti-5Cu and Ti-7Cu had strong bactericidal activity,and the overall antibacterial performance was Ti-7Cu?Ti-5Cu>Ti-3Cu.Ti-5Cu,Ti-7Cu could significantly inhibit the formation of bacterial biofilm.5.Ti-x Cu(x=3,5,7 wt.%)alloys exerted little effects on the proliferation of MC3T3-E1cells,and Ti-5Cu was the most potent for cell proliferation.At the same time,it was found that the RGR of Ti-x Cu alloys were all higher than 75%,which met the requirements of the implant material standard.According to the classification of toxicity evaluation standard(CTG),Ti-3Cu and Ti-5Cu were grade 0,Ti-7Cu was grade 1,Ti-3Cu and Ti-5Cu had no cytotoxicity.6.The addition of copper could promote the early osteogenic differentiation of MC3T3-E1 cells,and Ti-5Cu showed better bone-promoting ability.7.In vivo studies had found that Ti-5Cu implants had better antibacterial properties than Ti implants and could effectively reduce local symptoms such as gum inflammation.8.In vivo studies had shown that Ti-5Cu implants and Ti implants were similar to promote bone activity and bone integration performance.Based on the above research results,Ti-5Cu alloy is expected to become a new generation of oral metal implantation materials.