| Titanium metal and alloy have good biocompatibility and mechanical properties,and have been widely used in oral implant clinic.However,its surface has certain biological inertia,and its interface with surrounding bone shows non-specific binding,which affects the speed and quality of clinical bone integration.The study found that the surface modification of titanium implants can effectively improve its performance.As one of the surface modification methods of titanium,biological modification has become a research hotspot in this field.The surface of titanium implants can obtain better bone inductivity after biological modification.After being implanted into the body,it can actively react with the surrounding bone tissue,promote bone integration,shorten the clinical healing time,and achieve early good implant stability.The sustained-release system is to combine the bioactive factor with the sustained-release carrier on the surface of the implant through certain methods.When the implant is implanted in the body,the biological factor can be continuously released and its action time can be extended to promote the bone combination of the implant and surrounding tissues.At present,there are three main methods for the construction of bioactive molecular sustained-release systems on the surface of titanium implants: physical adsorption,chemical fixation and layer-by-layer self-assembly.Among them,layer-by-layer self-assembly(Lb L)is a relatively mature method in this field.Its basic principle is to alternately absorb polyelectrolytes with opposite charges on the surface of materials through electrostatic interaction,The self-assembled polyelectrolyte composite multilayer film is formed,and the multilayer film structure can be used as the sustained-release carrier of biological factors.Based on this,in this study,sodium alginate and chitosan with good biocompatibility were alternately adsorbed and deposited on the surface of titanium implants through Lb L technology,forming polyelectrolyte multilayer membrane structure and compounded with osteoblast-specific polypeptides to build its slow-release system,evaluate its slow-release effect,and provide new ideas and methods for the research on the surface biological modification of titanium implants.The details are as follows:The first part: alkali heat treatment of titanium surfaceThe purity of titanium substrate used in this experiment is 99.99%.First,it is processed into a round titanium sheet with a diameter of 10 mm and a thickness of 0.3mm;Then,use emery paper with different roughness(400,600,800,1000 mesh)to polish the sample;Then put the above titanium plates into glassware containing acetone,75% absolute ethanol and deionized water in turn,and wash them with ultrasonic for 20 minutes each.After drying,put 5.0mol/L sodium hydroxide solution into Na OH solution at 60 ℃ at constant temperature for 24 hours,and wash with deionized water and dry at low temperature to obtain alkali treated titanium;Then heat the alkali treated titanium sheet in the air to 200 ℃ for 2 hours and cool it naturally to obtain the alkali heat treated titanium sheet.The porous structure was prepared on the surface of titanium sheet,and the surface morphology of the sample was observed by field emission scanning electron microscope.The results showed that the surface of the sample prepared in this experiment was rough and porous compared with the mirror surface of smooth titanium sheet.The second part: PEC multilayer structure assembled on titanium surfaceThrough layer-by-layer self-assembly,sodium alginate and chitosan are alternately adsorbed and deposited on the surface of titanium after alkali heat treatment to form a multilayer polymer film structure.The samples were analyzed by field emission scanning electron microscope,contact angle measuring instrument,X-ray photoelectron spectroscopy and Fourier transform infrared spectrometer to test the preparation effect of alginate-chitosan multilayer film.The results of field emission scanning electron microscopy showed that the titanium surface was porous after the construction of the multilayer film structure,and some of the pores were filled,forming an uneven membrane structure,which was enlarged to(60.0k times)and showed a similar three-dimensional network structure.The contact angle experiment shows that the number of contact angles of the surface after alkali heat treatment is significantly reduced compared with that of pure titanium,and the hydrophilicity is enhanced.The hydrophilicity of the surface of the sample after sodium alginate-chitosan coating is decreased,but it is still improved compared with that of pure titanium.X-ray photoelectron spectroscopy showed that the Ti peak intensity weakened and N peak appeared after loading the coating,which proved that the alginate-chitosan multilayer film was successfully deposited on the surface of the sample.Fourier transform infrared spectroscopy showed that the absorption peaks appeared at 1620 cm-1 and 1403 cm-1 after the multilayer film was assembled on the titanium surface,which were the characteristic peaks of sodium alginate and chitosan,indicating that the chitosan-sodium alginate multilayer structure had been successfully constructed on the titanium surface.The third part: Preparation and sustained release detection of compound specific peptide sustained release structureIn the experiment,10-5 mol/L osteoblast-specific recognition polypeptide solution was prepared first,and then the three groups of samples were immersed in the polypeptide solution for 2 hours and then taken out.X-ray photoelectron spectrometer detects the element composition of the material surface,Fourier infrared spectrometer detects the functional groups of the material surface,fluorescence microscope shows the specific polypeptide composition of the material composite,and then ultraviolet spectrophotometer detects the content of the specific recognition polypeptide of the osteoblast on the sample surface and draws its release curve.X-ray photoelectron spectroscopy showed that at 400 e V,the N1 s peak of the peptide modified group increased,indicating that the specific peptide has been successfully loaded into the chitosan-alginate polymer multilayer.Fourier transform infrared spectroscopy showed that after the adsorption of the specific polypeptide,the wave peak at the absorption wave of 1630 cm-1 was basically consistent with the standard polypeptide,which confirmed that the specific polypeptide had been successfully carried on the surface of titanium substrate.Different amounts of fluorescence can be seen on the surface of the three groups of materials,and the amount of fluorescence on the surface of the chitosan-alginate polymer multilayer film group is the largest and uniform,which proves that the target peptide has been successfully fixed on the surface of titanium.The polypeptide release curve shows that the chitosan-sodium alginate multilayer film group has the longest polypeptide release process,and the release amount continues to increase until the 7th day,and the release effect is more ideal than the other two groups.The fourth part: evaluation of biological effects of specific polypeptides carried on titanium substrate on human skull osteoblastsAfter the recovery of human skull osteoblasts,the cells were subcultured to the third generation,and the cell density was adjusted.The cells were inoculated on the surface of smooth titanium sheets after high-temperature sterilization and ultraviolet sterilization,alkali heat treatment,and chitosan-sodium alginate multilayer film modified titanium sheets.The cells were stained and fixed,and the cell adhesion on the titanium surface of different experimental groups was detected with an upright fluorescence microscope.The MTT test was performed on the cells of each group,and the OD value of each group was measured,The effects of different materials on the adhesion and proliferation of human skull osteoblasts were compared.The cell experiment showed that compared with the other two groups of samples,the surface of the chitosan-alginate multilayer film group with composite specific polypeptide had a better promoting effect on the adhesion and proliferation of human skull osteoblasts.In this study,the electrostatic self-assembly technology was used to construct e xtracellular matrix on the surface of activated(alkali heat treated)titanium imp lants.Sodium alginate and chitosan were alternately adsorbed and deposited onthe surface of titanium implants to form a multilayer membrane structure.Ost eoblast-specific recognition peptides were compounded into the multilayer mem brane structure,and a sustained release system of osteoblast-specific peptides w as constructed on the surface of titanium,Thus,the amount of biological coati ng on titanium surface can be controlled and the specific polypeptide can be r eleased slowly.Cytological experiments showed that the sodium alginate and c hitosan multilayers loaded with specific peptides showed good affinity for hum an skull osteoblasts,which could significantly promote their adhesion and prolif eration on the surface of implants.This study provides a new idea and method for the study of the surface biological modification of titanium implants. |
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