Osteogenic Expression And Antibacterial Properties Of Copper Core-carbon Shell Nanoparticle Composite Coating On Titanium Surface

Objective:Implants and micro-implants have become the more common restorative materials used in clinical work in dentistry.Aseptic loosening and endophytic infection are the two most common causes of implant loosening and dislodgement.Titanium and its alloys（Titanium,Ti）have superior mechanical properties and good biocompatibility as biomedical materials.However,the bond between the titanium implant and the surrounding bone tissue is only mechanical and does not have antimicrobial properties,which makes it prone to loosening and dislodging.Surface modification technology can give special biological functions to titanium while retaining its original excellent properties.This experiment aims to prepare copper core-carbon shell nanoparticle coating on pure titanium surface,evaluate its biocompatibility,osteogenic ability and antibacterial activity,and provide a theoretical basis for the development of suitable implants for patients.Methods:1.Using the method of plasma arc evaporation of pure copper ingot method to prepare copper core-carbon shell nanoparticles,and observe the morphological structure and physical composition of the material by scanning electron microscopy（SEM）,high-resolution transmission electron microscopy（HRTEM）and X-ray diffraction（XRD）.2.After high-temperature shell-breaking treatment of the nanocapsules,the surface of pure titanium samples was sandblasted acid etched and alkali heat treated（SLA+AHT）.The nanoparticles were spin-coated on the surface of the titanium sheet and the coating morphology was observed by SEM.3.Bone marrow mesenchymal stem cells（BMSC）were selected as the experimental cells,and the cell proliferation ability and cytotoxicity of the material were evaluated by CCK-8 method;the calcified nodules of bone formation were observed by alizarin red staining;the m RNA expression levels of the genes Runx2,OPN and COL-1 related to cell osteogenesis were determined by Q-PCR.4.The nanoparticle coating material with the strongest osteogenic ability was selected and placed in a constant temperature incubator at 37°C,soaked in artificial saliva for 1,3 and 7 d.The amount of Cu²⁺dissolved in the leachate was detected by inductively coupled plasma mass spectrometry（ICP-MS）.5.The antibacterial activity of the nanoparticle coating was evaluated by plate counting.The bactericidal effect of the material was evaluated by plate counting method;laser confocal microscopy（CLSM）live-dead staining analysis was performed to compare the content of live-dead bacteria on the surface of the material;SEM was used to observe and analyze the surface morphology of the biofilm.Results:1.The synthesized Cu@C nanocapsules were nearly spherical in shape with smooth surface by SEM,and the typical shell-core structure was identified as graphitic carbon by HRTEM,and the core of the synthesized nanocapsules was determined to be Cu by XRD patterns,indicating the successful preparation of Cu-core carbon-shell nanoparticles.2.The results of CCK-8 showed that 5Cu@C-Ti had the strongest ability to promote the proliferation of BMSC when co-cultured with BMSC for 3,5 and 7 d.The cytotoxicity of 3Cu@C-Ti and 5Cu@C-Ti were both grade 0,and the cytotoxicity of7Cu@C-Ti was grade 1;the results of alizarin red staining showed that all groups had mineralized nodule formation,indicating that the material can induce osteogenic differentiation of BMSC cells,and the A562 nm absorbance results showed that the5Cu@C-Ti group had the highest degree of mineralization;the detection of osteogenic performance-related factors revealed that the gene expression levels of Runx2,OPN and COL-1 were higher in the 5Cu@C-Ti group than in the 3Cu@C-Ti,7Cu@C-Ti and control groups.4.The results of immersion experiments showed that 5Cu@C-Ti was effective in preventing sudden release and could be released continuously during the measured time period.5.The results of plate counting experiments showed that the5Cu@C-Ti surface had excellent antibacterial properties compared with the control group Ti,and the antibacterial rate reached over 99.9%.The live-dead staining results showed that almost all of the 5Cu@C-Ti surface was dead bacteria with ruptured cell membranes,further indicating that the 5Cu@C-Ti surface had good antibacterial performance.The scanning electron microscopy results showed that a large number of Staphylococcus aureus adhered to the Ti surface,and the bacteria were spherical in shape,and a large number of bacteria gathered and stacked into clusters in a grape bunch arrangement.The surface of the material showed adherent broken and dead bacteria,and the bacteria appeared to be obviously collapsed and crumpled.Conclusion:1.copper-core-carbon-shell nanoparticles were successfully prepared and copper-core-carbon-shell nanoparticle coatings were prepared on the titanium surface.2.all three groups of Cu@C-Ti materials met the requirements of implant material standards.According to the cytotoxicity evaluation criteria,3Cu@C-Ti and 5Cu@C-Ti were graded as grade 0,7Cu@C-Ti was graded as grade 1 and 5Cu@C-Ti had the best comprehensive osteogenic ability.3.5Cu@C-Ti can stably release Cu²⁺and has strong bactericidal activity on the surface,which can well inhibit the formation of Staphylococcus aureus biofilm.