| ObjectiveTitanium and its alloys have excellent mechanical properties,corrosion resistance and biocompatibility which are the preferred choice for dental implant materials.However,the dense oxide layer on the surface of the titanium makes the material biologically inert,resulting in slower bone integration after implantation in the body;in addition,the surface does not have antibacterial properties,it easily adheres to bacteria and forms biofilm,thereby causing infection or peri-implantitis,even leading to treatment failure.Therefore,improving osseointegration and imparting antibacterial properties to the surface are of great value to the implant application.In many titanium surface modification methods,alkali heat treatment can affect its biological properties by regulating surface morphology,chemical composition,hydrophilicity and has the characteristics of simple operation and easy control.It is possible to construct a hierarchical porous structure composed of micron-scale and nano-scale pores,and the surface is rich in hydroxyl groups and has super-hydrophilic properties,which lays a good foundation for subsequent biomolecule modification.The dopamine molecules that adhere to marine mussels have catechol and amino groups,which can 1)self-polymerize to form polydopamine with more reactive groups,2)firmly adhere to the surface of many materials to improve biocompatibility,3)realize the bridging of other biomolecules on the surface of the material;4)chelate or even reduce metal ions.These properties make it an important choice for implant surface modification.Among the broad-spectrum antibacterial agents,silver has the advantages of strong antibacterial properties,high stability,and no bacterial resistance.Compared with traditional silver ions,nano-scale silver has a stronger antibacterial properties due to its small size and large specific surface area.In this research,the surface of pure titanium was modified to make full use of the bearing capacity of porous structure and the reducing ability of dopamine.In this research,the surface modification of pure titanium was operated to make full use of the bearing capacity of porous structure and the reducing ability of dopamine.It is expected to construct a multifunctional coating with good bone integration and antibacterial properties to accelerate the osseointegration of implants.At the same time,preventing the occurrence of peri-implantitis and improve the success rate of implant.In addition,this research made a detailed comparison of the performance of polydopamine-nanosilver coatings on smooth and porous surfaces,providing theoretical and technical support for implant design.Methods(1)The porous morphology was constructed on smooth titanium surface by alkali heat treatment,then polydopamine coating was deposited on porous titanium surface by aqueous dopamine deposition,and then immersed in silver nitrate solution with different pH values.Polydopamine-nanosilver coating was constructed on porous titanium surface in different acid-base environments by using the reduction ability of dopamine.Physical and chemical properties were characterized by Field emission scanning electron microscopy(FE-SEM),Energy dispersive spectrometer(EDS)and X-ray photoelectron spectroscopy(XPS).The effects of different acid-base environments on the construction of dopamine-mediated nano-silver coatings were evaluated.(2)Silver ions can also be reduced to nano-silver on the smooth titanium surface by the same method in suitable acid-base environment.The physical and chemical properties and surface wettability of the samples constructed on the smooth surface and the porous surface were compared by FE-SEM,XPS,and contact angle tester.(3)The release behavior of silver ions from polydopamine-nano-silver coatings on smooth titanium surface and porous titanium surface in mineralization solution was analyzed by inductively coupled plasma optical emission spectrometry(ICP-OES),and the release behavior of silver ions from porous nano-silver coatings in mineralization solution and deionized water was also analyzed.(4)The antibacterial properties of the samples against six common oral micro-organisms were examined by fluorescent staining of hve/dead bacteria,zone of inhibition test,and turbidity method(microbial proliferation curve),Including Gram-negative bacteria E.coli and Gram-positive bacteria:S.aureus,S.mutans,A.israelii,L acidophilus and C.albicans.(5)By immersing the samples in biomimetic mineralization solution and simulating the changes of surface properties after implantation,the morphology and chemical composition of the samples after mineralization were analyzed by FE-SEM and XPS,and whether the coatings had the potential to promote non-cell-mediated osteogenesis on the implant surface was analyzed.(6)By actin fluorescence staining,MTT assay and alkaline phosphatase(ALP)activity,the effects of the constructed surface and mineralized surface of nano-silver coating on mouse embryonic osteoblasts(MC3T3-E1)were analyzed,and the cell-mediated osteogenic potential of the sample surface was evaluated.Results:(1)The results of FE-SEM,EDS and XPS showed that the pH values of silver nitrate solution affected the construction of dopamine-mediated nanosilver coatings.Compared with acidic and neutral environments(pH4,pH7),alkaline environment(pH 10)was more conducive to the deposition of nanosilver,resulting in more nano-silver particles,smaller size and more uniform distribution-(2)After alkali heat treatment,a large number of open micro-nano porous structures were formed on the surface of smooth titanium.The pore wall was a large number of pillar-like structures.The wall thickness of the pillar was about 30-50nm.Subsequently,polydopamine coatings and polydopamine-nanosilver coatings were successfully constructed on both micro-nanoporous and smooth surfaces.Compared with the polydopamine-nano-silver coatings on smooth surfaces,the coatings on porous surfaces obtained more nano-silver particles with smaller size(20-40nm)and more uniform shape.(3)The water contact angle of porous surface after alkali heat treatment was 25.3±4.1°,the hydrophilicity was significantly improved compared with the smooth surface(79.0±6.4°).On the smooth surface,the contact angle of dopamine modified porous surface was 66.5±3.4°,the hydrophilicity was improved,but the nano-silver made the surface hydrophobic,the contact angle increased slightly to 72.3±3.5°;On the porous surface,polydopamine and silver nanoparticles affected the surface hydrophilicity successively,and the water contact angle was increased to 50.2±5.4°and 76.5±6.7°respectively.(4)ICP-OES results showed that polydopamine-nanosilver coatings on smooth surfaces exhibited a slow decline and a high dose of silver release in mineralization solution,while porous nanosilver coatings exhibited a high dose release in the initial stage,followed by a low dose release in the mineralized solution·After 30 days,there was still 0.11±0.05μg and 0.09±0.02μg silver release in the smooth surface coatings and the porous surface coatings,showing good persistence;The porous nano-silver coatings exhibited stable and high-dose silver release in deionized water,and the cumulative release curve was approximately linear,which was significantly different from that in mineralization solution.(5)Fluorescence staining of living and dead bacteria on the surface of samples showed that the porous surfaces after alkali heat treatment were more susceptible to microbial adhesion than the smooth surfaces.After nano-silver modification,the adherence of six common oral microorganisms were inhibited on both the porous coating surfaces and the smooth coating surfaces.ZIO experiment showed that the surface of nano-silver modified coating have manifested obvious and transparent zone of inhibition,the both two coatings can inhibit microbial growth in the surrounding environment to a certain extent.Turbidimetric results showed that the two coatings modified by nano-silver completely inhibit microbial growth in liquid environment,and have obtained good and stable antibacterial properties.(6)The results of FE-SEM and XPS after mineralization experiments showed that the porous surface after alkali heat treatment can form more mineralized products than the smooth surface;the surface of polydopamine coating had the most mineralized products;the mineralized products of polydopamine-nanosilver coating on porous surface were more than that of polydopamine-nanosilver coating on smooth surface,suggesting that the porous nanosilver coating had higher non-cell-mediated osteogenic potential.The mineralization degree of different coatings was related to the surface structure and the amount of polydopamine exposed on the surface.(7)The results of actin fluorescence staining showed that the modification of nano-silver delayed the adhesion of osteoblasts,but with the prolongation of culture time,the adhesion and spreading of osteoblasts on the surface of nano-silver coating improved significantly,and the activities of MTT and ALP also increased.At the corresponding time point,the number and morphology of osteoblasts adhering to dopamine-mediated mineralized coatings were better than those of non-mineralized coatings,and the activities of MTT and ALP were also higher than those of non-mineralized nano-silver coatings.In addition,compared with the smooth structure of nano-silver coating mineralized surface,the porous structure of nano-silver coating mineralized surface was more conducive to the adhesion,proliferation and differentiation of osteoblasts,showing a better potential for cell-mediated osteogenesis.Conclusion and significanceIn this research,polydopamine-nanosilver coatings were successfully constructed on micro-nanoporous or smooth titanium surface by alkali heat treatment aqueous dopamine deposition and silver nitrate solution immersion.Compared with acidic and neutral environments,silver nitrate solution in alkaline environment was more conducive to dopamine-mediated nano-silver deposition;Compared with nano-silver coating on smooth surface,more and smaller nano-silver particles were obtained on porous surface,and more dopamine was exposed on porous surface,which induced more mineralization.This led to the rapid release of silver on porous surface at the initial stage and the rapid reduction of silver release at the later stage.The nano-silver coatings constructed on both surfaces significantly inhibited the adhesion and proliferation of microorganisms,and there was no significant difference in antibacterial properties.It was proved that the modified coatings solved the problem of infection on porous surfaces,but the nano-silver coatings delayed the adhesion and proliferation of osteoblasts,while dopamine-mediated mineralization improved the compatibility of osteoblasts,especially on porous surfaces.Therefore,It is speculated that the nano-silver coatings on porous surfaces can exert superior antibacterial properties and reduce the risk of infection at the initial stage after implantation.With the gradual absorption of calcium and phosphorus from body fluids,mineralized layer will be formed rapidly after implantation,which further improves cell compatibility and promotes osteogenic differentiation,thus achieving osseointegration,which fulfils the requirements in vivo and has potential for clinical application. |
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