Establishing Antibacterial Multilayer Films On The Surface Of Direct Metal Laser Sintered Titanium Primed With Phase-transited Lysozyme

ObjectivesWith the rapid development of digital technology revolution,3D printing technology has been increasingly become popular in the field of medicine.Direct metal laser sintering is a technology that allows the fabrication of titanium(Ti)implants with a functional gradation of porosity and surface roughness according to three-dimensional(3D)computer data.The hardness and elastic modulus of the porous titanium implant are very close to that of natural bone tissue,and the stress conduction is similar to that of the body.At the same time,the rough pore structure provides specific site for new bone ingrowth,The osseointegration strength and stability of the implant were enhanced,and the interconnected pore surface microstructure is more conducive to the transport of nutrients and nutrients.However,the surface roughness of direct metal laser sintered titanium(DMLS-Ti)implants may provide abundant binding sites for bacteria.Bacterial colonization and subsequent bioflm formation can cause unsatisfactory cell adhesion and implant-related infections.At the same time it will also have an impact on cell adhesion,proliferation.To prevent such infections,a novel phase-transited lysozyme(PTL)was utilized as an initial functional layer to simply and effectively prime DMLS-Ti surfaces for subsequent coating with antibacterial multilayers.Methods1 To produce a diameter of 10 mm micro-roughness of the implant surface by direct metal laser sintering,the titanium surface coating construction with activation phase transition of lysozyme,then on the titanium surface with minocycline-loaded polyelectrolyte multilayers of hyaluronic acid(HA)and chitosan(CS)formed via a layer-by-layer(LBL)self-assembly technique on PTL-functionalized DMLS-Ti Using field emission scanning electron microscopy(FE-SEM)observation of surface morphology of self-assembly coating;X-ray photoelectron spectroscopy(XPS)analysis of the composition of each assembly coating elements;contact angle measuring instrument for measuring the sample hydrophilicity/hydrophobicity;nano-indentation test the mechanical properties of sample surface.2 To evaluate antibacterial effect of antibacterial coating by plate count method with Streptococcus gordonii(S.gordonii)and Streptococcus sanguis(S.sanguis)in vitro;the activity of adherent bacteria on the surface of the sample after SYTO 9/PI fluorescence staining was observed by confocal laser scanning microscope.3 To evaluation of minocycline-loaded antibacterial multilayers biocompatibility by MC3T3-E1.Lactate dehydrogenase kit was used to detect the cytotoxicity of the modified lysozyme coating and self assembled antibacterial coating;the morphology and quantity of cell adhesion on the surface of the samples were observed by confocal laser scanning microscope;CCK-8 kit was used to characterize the cell proliferation.The osteogenic differentiation of the cells with different surface structure was detected by alkaline phosphatase kit.Results1 DMLS-Ti were successfully produced,the results of FE-SEM showed that the surface morphology of DMLS-Ti with the uniform distribution of phase-transited lysozyme modified coating and self-assembled coatings were observed.The results of XPS showed that the phase-transited lysozyme modified coating and the self-assembled coatings were successfully deposited on the surface of titanium.The contact angle experiment showed that the hydrophilicity of the modified samples was improved obviously.The results of nano-indentation showed that the coating reduces the hardness and elastic modulus of the sample at a certain extent.2 The experiment showed that the antibacterial effect of the minocycline-loaded polyelectrolyte antibacterial coating was achieved the experimental results of colony count assay and confocal laser scanning microscopy indicated the minocycline-loaded polyelectrolyte antibacterial coating can effectively inhibit the bacterial biofilm.3 The cytotoxicity showed that phase-transited lysozyme modified coating and self-assembled coatings had no toxicity,which could promote the early adhesion,proliferation and osteogenic differentiation of mouse osteoblasts.It show good biological activity.ConclusionsThe experimental results indicate that the minocycline-loaded polyelectrolyte multilayers of hyaluronic acid(HA)and chitosan(CS)formed via a layer-by-layer(LBL)self-assembly technique on PTL-functionalized DMLS-Ti were designed to inhibit pathogenic microbial infections while allowing the DMLS-Ti itself and the modifed coatings to retain acceptable biocompatibility.Due to the simple operation,low cost and high safety,the phase-transited lysozyme will provide a new method for the modification of the surface.At the same time,the experimental results showed that the layer by layer self-assembly technique was also suitable for the direct metal laser sintering of titanium.This design is expected to gain considerable interest in the medical feld and to have good potential for applications in multifunctional DMLS-Ti implants.