The Experimental Study Of Immobilization Of The Peptide That Specifically On The TiO₂ Nanotubes In Vitro

Pure titanium and its alloys have been widely used because of their excelle nt biological and mechanical properties.However,the titanium substrate belongs t o the biological inert surface and many disadvantages such as long bone healing period and poor effect after implantation.The TiO2 nanotubes can be used suita ble carrier as biological active molecules to modify the material surface due to it s large surface area,regular structure.While achieving structural and functional m odification synergistic effect to improve the biocompatibility of materials.This stu dy aims to covalently bonding to the TiO₂ nanotubes by polydopamine,which the TiO₂ nanotubes were made by Anodic oxidation.eventually,titanium nanotubes-do pamine-peptide composite bioactive layer was built successfully.Osteoblasts were cultured with the bioactive layer in vitro to evaluate the impact on the adhesion and proliferation.In order to provide a new research approach and experimental f oundation of the biolization modification on the surface of titanium implant.PartⅠ The preparation of The titanium nanotube arraysThe titanium plate was processed into thickness 0.2mm,diameter of circular titanium 1mm.The titanium plate was polished to mirror shape by metallographi c,then be cleaned ultrasonically ethanol,deionized water in turn 2 times,each ti me for 10 minutes in acetone and be spare after the dry.The smooth titanium pl ates were immersed into ethylene glycol electrolyte containing 90mmol/L ammoni um fluoride with titanium as anode and graphite as cathode.Anodic oxidation div ided into two steps:6V,10min;45V,50min.The samples were taken out after cl eaning ultrasonically 2 times,each time for 5 minutes by glycol and deionized water and be spare after the dry.The samples were characterized by FESEM.FESEM showed that the surface of the titanium plate is like mirror shape,and the sample of the titanium nanotube group is like honeycomb tubular structure,and the size of the lumen is uniform,which the diameter of the pipe is 50 to 90nm and the diameter of 70nm is about 2/3.Part Ⅱ The immobilization of the peptide that specifically bind to osteoblasts on the surface of titanium nanotubesThe titanium nanotubes specimens were completely immersed in the concentr ation of dopamine 2mg/ml solution（Tris buffer concentration is 10mmol/l,PH=8.5）for 12h in the light treatment,which made that The surface of the titanium sub strate is formed with a layer of dopamine film that is rich in catechol group.The dopamine modified titanium nanotubes samples were completely immersed in th e concentration of 10^-5mol/L solution of the peptide that specifically bind to oste oblasts the specific with being FTIC marked（Tris buffer concentration is 10mmol/l,PH=8.5）for 12h in the light treatment.The catechol group is further oxidized t o quinone in the alkaline environment,which can result in chemical bond with a mino and carboxyl of peptide in functional areas of peptide to immobilize pepti de on the titanium substrate surface.The samples were characterized by field-emis sion scanning electron microscopy,atomic force microscope,contact angle measur ement,X-ray photoelectron spectroscopy and immunofluorescence,which test samp le properties and demonstrated that the peptide that specifically bind to osteoblast s was successfully bonded to the TiO₂ nanotubes.The AFM results showed that Comparation with the group of smooth titanium and titanium nanotubes,the peptide modified group molecular particles are evenly distributeduniform,relatively flat,which 3D honeycomb has changed distinctly and the experimental group surface roughness has increased.Contact angle results indicated contactanglebecamesmalleraftercontinuousmodificationbyanodic oxidation,dopamine and peptide,which demonstrated the hydrophilic properties of materials has increased.XPS results showed that the peptide modified group substrate surface increased significantly in the 400eV nitrogen peak,which indicated that the peptide molecules containing nitrogen element is attached to the surface of the substrate.Immunofluorescence showed that Comparation with the smooth titanium coating group,the number of fluorescent of the titanium nanotubes coating fluorescent group was significantly moreand uniform distribution the titanium nanotubes coating group,which confirmed that the osteoblast specific peptides were successfully immobilized on the titanium substrate and titanium nanotubes can be used as a carrier of the peptide molecule.Part Ⅲ Effects of peptide modified titanium substrates on the biological behavior of osteoblasts.The human calvarial osteoblasts were seeded on smooth titanium plates,titanium nanotubes.The number of cells were counted on the different surface of the titanium substrate by immunofluorescence and then determined by MTT.Single factor analysis of variance was used to evaluate the effect of materials on the adhesion and proliferation after arranging the experiment data.The results showed that compared with the other three groups of substrates,the surface of human calvarial osteoblasts has better performance of adhesion and proliferation on the surface of peptide modified substrate.Conclusion We prepared titanium nanotube substrate by using anodic oxidation method,then the peptide was covalently bonding to the TiO₂ nanotubes by polydopamine.Eventually,the titanium nanotubes-dopamine-peptide Composite biological bioactive layer was built on the TiO₂ nanotubes.The results showed that the layer had good affinity to human calvarial osteoblasts by cytological test.The study provides a new research approach and experimental foundation of the biolization modification on the surface of titanium implant.