Mussel-Inspired Deposition Of Copper On Titanium For Bacterial Inhibition And Enhanced Osseointegration In A Periprosthetic Infection Model

Part?Preparation of copper-coated titanium using polydopamine-assisted deposition techniqueObjective: The polydopamine(PDA)coating was formed by the self polymerization of dopamine and deposited on the titanium surface under the condition of weak alkaline,Copper ions were anchored on the surface of pure titanium(Ti)materials by chelation of polydopamine,then the titanium composite materials which surface loaded copper were prepared and characterized.Method:Prior to PDA coating,Ti disks and were first ultrasonically cleaned stepwise using acetone,ethanol and deionized water.Dopamine solutions were prepared by dissolving dopamine at a concentration of 2mg/m L in 10 m M Tris-HCl buffer solutions(p H=8.5).The Ti disks and were then soaked in the dopamine solution for overnight for PDA coating.Cu ions were immobilized in PDA coating via soaking method.Cu Cl2 was mixed with deionized water to prepare solutions with different Cu concentration of 0.02 mmol/L,0.1 mmol/L and 0.5 mmol/L.To observe the surface morphology of the different sample,Scanning electron microscopy(SEM)was used.The surface chemistry of the samples was examined qualitatively using X-ray photoelectron spectroscopy(XPS).Surface wettability was assessed by a tensiometer.The release amounts of copper ions from different Ti-PDA-Cu samples were measured within different immersion time of intervals using ICP-OES.Result: The surface morphology of the coatings was not significantly changed by the polydopamine coating and the doped copper element as indicated by scanning electron microscopy.The content of Cu was estimated to be 4.28 at %,6.01 at%,and 8.47 at% from the XPS analysis of Ti-PDA-Cu 0.02,Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5 samples,respectively.To evaluate wetting properties of different Ti samples,the water contact angles on the samples were measured.Compare with Ti substrates(80 ±6.76°),Ti-PDA,Ti-PDA-Cu 0.02,Ti-PDA-Cu 0.5,and Ti-PDA-Cu 0.1 samples all displayed higher hydrophilicity,with much lower(p<0.01)contact angles of 45 ± 2.81°,49 ± 4.38°,54 ± 4.38° andlower(p<0.05)contact angles of 60 ± 5.45°,respectively.The copper ion release test showed that the copper ions from the three Ti-PDA-Cu groups were able to release copper ions in the surrounding solution.Conclusion: Using the self polymerization of dopamine and the chelation of polydopamine,the copper coating with different content can be prepared on the surface of titanium.The polydopamine coating and the doped copper did not significantly change the surface morphology of the coating,but the chemical composition of the coating changed and the hydrophilicity of the coating improved significantly.Copper coatings with different contents could continuously release copper ions to the surrounding solution.Part ? In vitro antibacterial properties of copper-coated titanium surface using polydopamine-assisted deposition techniqueObjective: To investigate the antibacterial properties of titanium composite materials with different contents of copper against Gram positive Staphylococcus aureus and Gram negative Escherichia coli.Method: Pristine Ti sample served as the control and Ti-PDA,Ti-PDA-Cu 0.02,Ti-PDA-Cu 0.1,and Ti-PDA-Cu 0.5 samples constituted the experimental groups.Staphylococcus aureus strain(ATCC 25923)and Escherichia coli(ATCC 25922)were used as experimental bacterial strains.The strains of Staphylococcus aureus and Escherichia coli were inoculated in each group for 24 hours,counting the living bacteria by plate counting method and the antibacterial properties of all kinds of materials was observed.The experiment was repeated three times to calculate the antibacterial rate and take photos of the coated plates.Result: The results of antibacterial experiment by plate counting method suggest that the average antibacterial rate of against S.aureus on Ti-PDA-Cu 0.5 and Ti-PDA-Cu 0.1 are 99.4% and 91.9%,remarkably higher(p<0.01)than 41.5% and 10.2% of Ti-PDA-Cu 0.02 and Ti-PDA substrates.Similar tendency were observed for E.coli with the average antibacterial rates measured to be 11.9%,46.2%,92.3% and 98.3% for Ti-PDA,Ti-PDA-Cu 0.02,Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5,respectively.Photos of the coated plates clearly show that the number of bacterial colonies on plates decreases with increased Cu content on different Ti-PDA-Cu substrates.On Ti,Ti-PDA and Ti-PDA-Cu 0.02 samples,there are a large number of bacterial colonies.In contrast,much fewer colonies could be found on Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5 samples.Conclusion: Compare to pristine Ti,titanium composite materials with different contents of copper exhibited excellent antibacterial activity against both S.aureus and E.coli,the number of bacterial colonies on plates decreases with increased Cu content on different Ti-PDA-Cu substrates.Part ? Biocompatibility of copper-coated titanium surface using polydopamine-assisted deposition techniqueObjective: To study the influences of copper-coated titanium surface using polydopamine-assisted deposition technique on the adhesion and proliferation of MC3T3-E1 osteoblast.Method: Pristine Ti sample served as the control and Ti-PDA,Ti-PDA-Cu 0.02,Ti-PDA-Cu 0.1,and Ti-PDA-Cu 0.5 samples constituted the experimental groups.MC3T3-E1 osteoblasts were cultured on the surface of each group at the same density.The morphology of cells on surface of disks was observed by SEM after 24 h incubation.Cell viability assay was conducted by CCK-8 and detect the number of cells adhered to the surface of each group at 1?3?5 and 7 days.Cell morphology and cytoskeletal arrangement of each group were observed by fluorescent staining after 24 h incubation.Result: The SEM observed that the cells cultured on the Ti,Ti-PDA,and different Ti-PDA-Cu substrates display different morphologies after 24 h culturing.As can be clearly seen,the cells showed relatively round shape when cultured on Ti.However,they exhibited much flattened,scattered and irregular shape on the surfaces of Ti-PDA and different Ti-PDA-Cu substrates.The result of CCK-8 showed that the number of cells steadily increased with increasing culture time for all five groups.Cell proliferation on Ti substrates were superior beyond that on Ti-PDA and different Ti-PDA-Cu substrates for 1?3 and 5 days.However,after 7 days of culturing,no differences can be found in OD values among Ti?Ti-PDA and different Ti-PDA-Cu substrates.After 24 h incubation,cells adhered well on these substrates as indicated by the well-spreading cytoplasm and development of numerous lamellipodia and filopodia processes using fluorescent staining.The cells cultured on Ti substrates were not fully spread and showed relatively round morphology which seemed to lack microfilaments.However,on PDA and different Ti-PDA-Cu substrates,the cells exhibited much flattened and irregular morphology,indicating stronger cell adhesion on the samples.Conclusion: MC3T3-E1 cells cultured on copper-coated titanium surface using polydopamine-assisted deposition technique not only has good biocompatibility and but also promote osteoblast adhesion and spread on material surface.Part ? In vivo antibacterial properties and osseointegration performance of copper-coated titanium surface using polydopamine-assisted deposition techniqueObjective: In vivo,the antibacterial properties and osseointegration performance of copper-coated titanium surface using polydopamine-assisted deposition technique were investigated.Method: A new model of implant-related osteomyelitis in tibia of rats were used to evaluate periprosthetic infection.Pristine Ti sample served as the control and Ti-PDA,Ti-PDA-Cu 0.02,Ti-PDA-Cu 0.1,and Ti-PDA-Cu 0.5 samples constituted the experimental groups.We used forty male SD rats weighing from 300 to 350 g for all in vivo assays.Five groups of experimental animals,and eight in each group.To access the tibial medullary cavity,a hole was drilled using a circular drill(1.2mm diameter)to make a channel from the proximal tibial metaphysis into the medullary canal.For bacterial inoculation,20 ?l of S.aureus suspension with a concentration of 104 cfu/m L was injected into the medullary cavity with a microsyringe.Following inoculation,the sterile implants(1.2 mm diameter and 10 mm length)were inserted into the medullary cavity until beneath the growth plate.We studied the antibacterial effect and osseointegration performance of each group in vivo by Micro-CT and histological sections and immunohistochemical staining.Result: H&E staining suggest that numerous neutrophils emerged around the tibia medullary cavity in the Ti,Ti-PDA,and Ti-PDA-Cu 0.02 groups.Many chronic inflammatory cell infiltrations were also observed after 4 weeks.In contrast,no sign of infection could be observed in Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5 groups.Masson's trichrome staining suggest that at week 2,fibrous tissue was detected at the bone/implant interface in Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5 groups.After 4 weeks,a large amount of woven bone with diminishing fibrous tissue was observed at the bone/implant interface in Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5 groups.As comparison,new bone tissue could be hardly found for the other groups.TRAP staining indicate a typical signs of bone resorption could be illustrated by the accumulation of osteoclasts around the Ti,Ti-PDA,and Ti-PDA-Cu 0.02 groups at 2 weeks,while the number of osteoclasts decreased after 4weeks.In contrast,no sign of osteoclasts was observed for Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5 groups,suggesting negligible bone resorption occurred in these cases.The inoculated S.aureus was visualized using immunohistochemical analysis.Despite same initial amount,at 2 weeks after surgery,bacteria were evidently abundant in Ti,Ti-PDA,and Ti-PDA-Cu 0.02 groups,while just a trace number of bacteria was found in Ti-PDA-Cu 0.1 group and no bacteria was found for Ti-PDA-Cu 0.5.After 4weeks,bacteria infection could be evidently observed in Ti,Ti-PDA,and Ti-PDA-Cu 0.02 groups.However,no bacterial survival either in single organisms and in colonies could be detected in Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5 groups.What's more,at 4 weeks after surgery,the 3D reconstruction image of Micro-CT scan and quantitative analysis further verify the results of histological observation.Little bone was found around Ti and Ti-PDA implants.However,more bone could be observed around Ti-PDA-Cu 0.02 implant,and the highest amount of newly formed bone could be apparently seen in the images of Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5.The calculated bone volume(BV/TV)under the same threshold and volume interest(VOI)for CT scanning demonstrate that the Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5 rods exhibited the highest percentage of BV/TV.Conclusion: Animal models of periprosthetic infection indicate that Ti-PDA-Cu 0.1 and Ti-PDA-Cu 0.5groups have excellent antibacterial properties and can promote osseointegration,so it helps to prevent complications related to periprosthetic infection.