| As the social development and the progress of people’realization in mouth rehabilitation,more and more people comes to plant or restore their missing teeth. The key to success of implant denture is the formation of good bone after implantation. Professor Branemark founded in the1960s Bone combined with theory laid the foundation of modern oral planting of biology, The theory of bone combined is as following:living bone tissue combined with implant directly, without the fibrous tissue around the implant, and this combination must be able to withstand weight.The current commonly oral implant materials is mainly pure titanium, titanium alloys, and some composite materials. The advantages of pure titanium is qualitative light, for vibration damping force is large, low modulus of elasticity, simultaneously has characteristics about high hardness and ultimate tensile (zhang) strength, yield strength, fatigue strength and corrosion resistance of fatigue limit. so the application of pure titanium implant very extensive. Not only that, because of the advantages of titanium and titanium alloy, in recent years, the titanium alloy has been widely used in orthopaedic clinic, and widely used in the field of orthopaedic surgery oral medicine. But the titanium is the biological inert materials, require for a long time to heal in the clinical application, and tissue response performance for nonspecific and randomness after implanting into body.Therefore, trying to explore a new implant surface modification methods for new implants, There are great scientific significance and practical prospect of clinical application that to make sure high success rate of planting significantly and shorten the implant and the alveolar bone bone integration time and relieve the suffering of the patient.In nearly20years, titanium surface treatment method, factor inducing osteogenesis, bone morphogenic factor related genes and gene carrier, the carrier of the modifications, complex and titanium surface of building a successful method of combining became the research hot spot and focus. A large number of studies have shown that the initial stability of the titanium implant and the long-term success rate were significantly higher than no implant surface roughening treatment after dealing with the physical chemistry.The reason may have two aspects, firstly, the inorganic composition is plant on the surface of the body surface after dealing with the physical chemistry could promote the adhesion, proliferation and differentiation of osteoblast, so as to promote the integration of bone;Secondly, coarsening surface can increase the surface area of the implant greatly, improve mechanical embedded together, and increase the initial stability of the implant.With the deepening of the research, The body surface biological chemical modification research that introduce the function clear active substances to grow on the surface become hot topics in the study of implant gradually. Relative to other physical and chemical methods, this method directly using the function expression of osteoblasts promote the activity of molecular reaction to influence the organization, to promote the new bone formation and thus this method is compared with the traditional physical and chemical method is more direct and effective, is expected to get faster than inorganic surface of implant bone integration speed. Studies have shown that functional protein (protein fiber adhesion, BMP, cell adhesion short peptide RGD, etc.) can be introduced into the implant surface. HBMP2were known as growth factors because of the strong induced osteogenesis ability. HBMP2exists in human hard tissues widely as bone growth promoting factor. They mainly distribute in cytoplasm with osteogenesis tendency, including osteogenesis, into glaze and odontoblasts and into cartilage cells, etc. and they can induce osteogenesis, and induce undifferentiated mesenchymal cells into osteoblast. The activity of gene hBMP2is strongest in7hBMP members that can be clonedis. But hBMP2metabolise is so fast by the body that it is difficult to form a continuous function, despite the volume of protein is so big that affect the thickness of the implant surface. So, to choose a suitable carrier effective and controllable reactive molecules gene is introduced into the implant surface and keep its good biological activity,and control the release of the implant surface active molecules at the same time, in the proper period of cell proliferation differentiation and tissue reconstruction maintain the appropriate concentration, is that we should focus on the fundamental problem of implant surface bioactivity.Gene carrier can be divided into viral vector and non viral vector. The transfection efficiency of viral vector is higher than non viral vector, but there are some security problems, such as virus recombination, immunogenicity, and therefore the non viral vector systems for gene therapy with virus carrier is more secure.In the numerous non viral vector system, cationic polymer is a very promising carrier. Chitosan is a kind of biodegradable nanoparticles only with cationic carrier materials existing in the nature, with good biocompatibility, biodegradable and mucosal adhesive, especially suitable for package carrier proteins, peptides, such as vaccines and gene bioactive molecules. Now instead of liposomes and virus main kinds of carrier chitosan as a drug, protein and gene carrier has been widely studied.Chitosan can combine with the negatively charged DNA through the positive charge on the surface of molecules, prevent DNA enzyme (DNaseI) on the degradation of DNA, the effective concentration of DNA. Moreover, there are some advantage about the strong adhesion effect, side effects, rich source, and a small price.But the dis advantage about low targeting and low transfection efficiency of chitosan often limits its application.In order to improve its targeting and transfection efficiency, much research on the chitosan molecular peptide modified, in order to reach the purpose of improving its transfection efficiency.RGD peptide is a kind of short peptide that contains1-arginine glycine-aspartate (Arg Gly Asp) sequence in the widespread, it is recognition sites of the interaction between integrin and the ligand, and it can combine with11kinds of integrin specificity, and promote the growth of osteoblasts and bone tissue regeneration, and inhibit adhesion between substrate and osteoclast. RGD targeting peptide modified chitosan as a gene expression vector, its transfection efficiency is higher than pure chitosan as carrier obviously.At the same time, the implant surface with fixing short RGD peptides, can cause bone growth around implant, make the implant enhancement of tensile resistance.Therefore, the graft copolymerization between RGD and chitosan, can improve the performance of the chitosan carrier, and improve the implant surface properties, and promote the osteogenesis of titanium implants surface from the level of gene and peptide.Currently, the methods to fix or release of biological macromolecules including physical adsorption method, chemical method, layer upon layer self-assembly method and carrier method.The Layer-by-Layer self-assemble technique is applied maturely more and more, on the surface of the base material that charged by electrostatic interaction, alternately with opposite charge on the adsorption of polyelectrolyte, form self-assembled multilayer polyelectrolyte complexes thin film. This technology proposed and used firstly by Decher G in the1990s. LbL technology preparation conditions is simple and gentle, and it is not subject to the restrictions by base material. The film has high thermodynamic stability, the amount of coating can be control by changing the number of coating cycle, and can be loaded cytokines with slow-release function. However, the Layer-by-Layer self-assemble technique work through the electrostatic effect. Because of the small molecular weight of RGD peptide, the charge is very few, so the RGD on the surface of implant is less. The Layer-by-Layer self-assemble technique is introduced to implant surface RGD assembly method, and combined with chemical coupling to assemble RGD, it can obviously promote the RGD on the growing surface binding force and quantity, and this is the important progress about RGD assembly method on implant surface.The osteogenesis sarcoma cell line MG63is recognized as people osteoblast phenotype characteristics of osteoblast model. So this study have compared effect on MG63cells proliferation before and after the modification of titanium surface, and compared the plasmid DNA transfection and expression efficiency in the surface of pure titanium MG63the titanium plate between sandblasting and sintering treatment.Objective:1, To explore new implant surface modification method, and shorten the bone integration time between implant and the alveolar bone and reduce the patient’s pain.2, The chitosan is used as a viral vector, and BMP, cell adhesion peptide RGD is introduced into the titanium plate surface, and to increase the speed of bone integration.3, To compare transfection efficiency and expression differences in the titanium plate surface between sandblasting and sintering treatment with BMP-2gene expression vector in MG63cell.Methods:1, The pure titanium surface treat with acid and alkali after sand blasting or sintering, and detect its characterization.2, Choose hBMP-2cdna as activation factor, design and synthesis of small molecules targeting peptide RGD peptide, choose biological macromolecular chitosan with good bone induced, the RGD (Arg Gly Asp) short peptide and chitosan through acylation reaction form RGD/CS complex coupling, evaluate the element content with the composite element analyzer, authenticate RGD/CS complex with infrared spectrometer.3, Compound condensing RGD/CS/BMP-2cdna complex, the RGD/CS/BMP-2cdna complexes were characterized using atomic force microscopy.4, The RGD/CS/hBMP-2cDNA complex has been grafted to titanium surface with Layer-by-Layer self-assemble technique and chemical coupling technology, and the surface of titanium has been dyeing test with EB. 5, Cultivate the osteogenesis sarcoma MG-63cells titanium surface grafting with RGD/CS/BMP-2cdna complex, and test the growth condition of cells in sand blasting and sintering of two different processing titanium surface; and the transfection efficiency RGD/CS/BMP-2cdna complex in MG-63cell has been tested with fluorescent microscope.6, Test BMP-2gene expression level in MG-63cells of different processing titanium surface through the Real-Time PCR and Western blotting experiment.Results:1, Ehrough infrared spectrum detection showed that after coupling the RGD peptides, CS-RGD near1630cm-1amide I bring increased significantly, in the process of coupling generates more carbon oxygen double bond, confirmed the amidation of successful CS and RGD peptide coupling;Through the element analysis shows that after coupling the RGD peptides, sample N atom content proportion of CS-RGD, than pure CS is high, it further shows that CS graft RGD success.2, Enzyme digestion and electrophoresis experiment showed that the BMP-2gene electrophoresis stripe size in line with expectations;Plasmid sequencing results show that the process of building a carrier, BMP-2gene mutation, carrier build is successful.3, Electrophoresis experiment showed that the CS-RGD/bmp2cDNA in the N/P=2, CS-RGD and bmp2cDNA completely compound;Atomic force microscope detection showed that the N/P=2～50RGD-CS/bmp2cDNA complex particle size is137-259nm, Zeta potential between+25mV～+30mV, and atomic force microscopy results showed that the compound for the class, spherical and well distributed;4, Pure titanium after sintering treatment, detected by scanning electron microscopy (sem) test, it is uniformly distributed over the surface of "Ω cave."5, EB dyeing experiments showed that the grafting with RGD-CS/bmp2cDNA complex titanium plate, can be EB stain, illustrating RGD-CS/bmp2cDNA complex graft titanium plate experiment success; 6, After enzyme digestion, RGD/CS/BMP-2cdna complex grafted in titanium surface has been released from chitosan,and the sequencing experiment showed that there is no gene mutation about RGD/CS/BMP-2cdna complex after grafting.7, Cell transformation experiment shows that the parcel again after CS coupling RGD BMP2plasmid, the transfection efficiency in MG63cells obviously a pure CS parcel BMP2plasmid transfection efficiency is high;8, Scanning electron microscopy (sem) test show that the surface treated by oxidation of mixed acid solution form "Ω" in the shape of a pure titanium plate, is advantageous to the growth of cells on the surface to stick wall;Training in graft RGD-CS/bmp2cDNA complex titanium plate on the MG63cells, part of the Mosaic in "Ω" in the shape of a concave hole, greatly improving the cells of the titanium surface adsorption force;9, Fluorescence microscope observation found that training in graft RGD-CS/bmp2cDNA complex MG63cells on titanium plate, titanium surface BMP2plasmid effectively into MG63cells;10, Real-Time PCR and Western blotting experiment showed that MG63cells growing in titanium plate with graft RGD/CS/Bmp-2cdna complex expressed BMP2proteins, and the expression level is higher than the MG63cells training in titanium plate without modification significantly.In conclusion, this study showed that RGD can effectively improve the cell transfection efficiency, CS coating with short peptide RGD, can be used as a carrier of titanium implant surface plasmid packaging;Surface after RGD-CS/bmp2cDNA complex modification of titanium implant, is advantageous to the MG63cells adherent growth on the surface, to improve cells on the surface of titanium NianZhaoLi;Titanium surface BMP2protein expression vector into a MG63cells effectively, cultivate in graft RGD-CS/bmp2cDNA complex titanium plate on the excess of MG63cells can express BMP2protein. |
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