Application Of Gold Nanoparticles Loaded Polydopamine Coating Modified Graphene Oxide Composite Material In Bone Defect Repair

Background:With the continuous development of modern society,the incidence of bone defects caused by trauma,limb deformities,tumors and infectious diseases is also increasing.Large bone defects will cause great inconvenience to the daily life and social contact of patients,and place an enormous burden on mind,body and economy.At present,the clinical treatment method of bone defects mainly includes autogenous bone and allogeneic bone graft.However,these treatments have some limitations,such as limited number of donors,immune rejection,infection and pain caused by secondary surgery.In recent years,with the continuous development of material technology,more and more biodegradable materials with excellent biological activity have been prepared,and these materials have largely solved the problems existing in the traditional bone defect treatment method.Among these bone repair materials,PLGA and other synthetic materials have attracted the greatest attention.These materials have excellent biocompatibility and biodegradability,which can be completely degraded into CO₂ and H₂O in the human body,and avoiding the secondary operation caused by prosthesis removal.Another advantage of PLGA materials is the high controllability of physical and chemical properties.By adjusting the composition proportion,molecular weight and crystallinity of these polymers,the degradation rate of materials can be accurately adjusted.PLGA has been approved by the Food and Drug Administration(FDA).However,these synthetic materials still have some disadvantages when used as bone defect repair materials.For example,their mechanical strength can not meet the special needs of weight-bearing sites,and bone repair materials lack the necessary bone conduction and induction functions.In order to overcome the above shortcomings,various functional modification methods such as material blending,functional coating and chemical grafting were often used to improve the bone repair effect of the materials.Among the many functional modification methods,the most common method is to blend PLGA material with inorganic particles to prepare inorganic-organic composites.By this way,the composites not only have a improve mechanical strength and in degradation properties vivo,but also maintain the osteoinduction activity of bio-active nanoparticles.However,a perfect bone repair material should have good bio-compatibility,osteogenic activity,suitable mechanical properties and good antibacterial properties.Simple blending of nanoparticles may not improve all the functions of PLGA materials.Therefore,in order to maximize the bone repair ability of materials,inorganic particle composite method often needs to be combined with other surface modification methods to further improve the function of polymer materials.The aim of this study is to improve the biological properties of PLGA scaffolds by applying the bio-active nanoparticles blending and the surface functional coating,and achieve the purpose of bone defects rapid healing.Purpose:The purpose of this study is combine with L-lysine functionalized graphene oxide(Lys-g-GO)with the gold nanoparticles-polydopamine functional coating(Au NPs-PDA)to improve the biological activity of PLGA scaffolds,and the effects of these two functional modification methods on the properties of the scaffold were observed.We hope to prepare a bone tissue engineering scaffold material with excellent bone repair ability.Method:1: Amino acid functionalized graphene oxide(Lys-g-GO)was prepared by surface grafting L-lysine(Lys),and PLGA/Lys-g-GO inorganic-organic composites were prepared by blending Lys-g-GO with PLGA.The effects of GO and Lys-g-GO nanoparticles on the physical and chemical properties of PLGA(surface morphology,mechanical strength and surface hydrophilicity,etc.)were detected by scanning electron microscopy(SEM),atomic force microscopy(AFM),contact angle tester and mechanical tester.BCA assay was used to detect the protein adsorption capacity of composite materials.MTT and fluorescence staining were used to detect the proliferation and adhesion ability of MC3T3-E1 cells on the composite material.Alkaline phosphatase activity(ALP)and alizarin red staining(ARS)were used to evaluate the effect of composite materials on cell osteogenic differentiation.To explore the effect of amino acid functionalized GO nanoparticles on the osteogenic induction ability of PLGA materials,so as to prepare for subsequent studies.2: Using PLGA/Lys-g-GO composite materials as the matrix materials,the composite porous scaffolds were prepared by phase conversion/particle leaching method.Gold nanoparticles(Au NPs)were deposited on the surface of the composite scaffold using polydopamine reductive properties,and prepare Au NPs-PDA@PLGA/Lys-g-GO scaffold materials.Then,the physical and chemical properties of different scaffolds were detected by SEM,contact angle tester and mechanical tester.Staphylococcus aureus and Escherichia coli were co-cultured with different scaffolds to detect the antibacterial properties of scaffolds.MTT,ALP activity,ARS staining and RT-PCR were used to detect the effects of Lys-g-GO nanoparticles combined with Au NPs-PDA functionalized coating on cell proliferation,adhesion and osteogenic differentiation.3: A model of rabbit radius defect was established.The osteogenic ability of different scaffold materials in vivo was detected by digital X-ray imaging,CT and Lane-Sandhu X-ray score.The regeneration of bone tissue was observed by HE,Masson and Sirius red staining.The bio-safety of different scaffolds was verified by observing the HE staining sections of experimental animals organs.The above experiments further verified the combined effect of Lys-g-GO nanoparticles and Au NPs-PDA functionalized coating on the biological properties of scaffolds,and investigated the potential value of Au NPs-PDA@PLGA/Lys-g-GO bone tissue engineering scaffolds in the application of bone defect repair.Result:1: By fourier transform infrared spectroscopy(FITR)and transmission electron microscopy(TEM)analysis,Lys was successfully grafted onto GO surface.Compared with unmodified GO,Lys-g-GO nanoparticles can improve the protein adsorption capacity and hydrophilicity of PLGA materials to a greater extent,but have no significant effect on the mechanical properties of the materials.Through cell experiments,we found that Lys-g-GO could significantly enhance cell proliferation,adhesion and osteogenesis differentiation.By blending Lys-g-GO,the biological activity of PLGA material was improved to a greater extent.2: Using phase conversion/particle leaching method,we successfully prepared a PLGA/ Lys-g-GO composite porous scaffold with good pore penetration structure.X-ray electron spectroscopy(XPS)and X-ray diffractometry(XRD)results showed that Au NPs nanoparticles were successfully loaded on the surface of the scaffold by polydopamine.The mechanical compression tests showed that Au NPs-PDA functionalized coating had no significant damage to the natural mechanical properties of the scaffold,and could significantly enhance the hydrophilicity of the scaffold.The antibacterial results showed that the combination of Au NPs-PDA functionalized coating and Lys-g-GO could greatly improve the antibacterial activity of PLGA scaffolds.Through in vitro cell experiments,Lys-g-GO and Au NPs-PDA coatings exhibited synergistic effects in promoting cell proliferation,adhesion,osteogenic differentiation and calcium deposition.The osteogenic induction of Au NPs-PDA@PLGA/Lys-g-GO scaffolds were better than those of other experimental groups.3: Through the experiment of repairing the radius defect in rabbits,the results of CT and X-ray results showed that the Lys-g-GO and Au NPs-PDA coatings significantly improved the bone repair rate of PLGA scaffolds.The results of HE,Masson and Sirius red staining showed that the Au NPs-PDA@PLGA/Lys-g-GO group had a large amount of new bone formation and a high amount and maturity of collagen fibers.The HE staining results of the experimental animals organs showed that scaffold materials was not bio-toxic,and had good biocompatibility.Conclusion:The results of this study showed that the modification methods of Lys-g-GO nanoparticles and Au NPs-PDA surface functionalized coating could effectively improve the biological activity of PLGA scaffolds.Two modification methods could make up for each other to a certain extent,and had a significant synergistic effect in improving the bone repair ability of scaffold materials.Au NPs-PDA@PLGA/Lys-g-GO scaffold material has good hydrophilicity,mechanical strength and antibacterial properties.In vitro,scaffold materials can significantly improve the cell behaviors of osteoblasts such as adhesion,proliferation,osteoblastic differentiation and calcium deposition.In vivo,scaffold materials can promote the formation of new bone and collagen matrix.Thus,Au NPs-PDA@PLGA/Lys-g-GO scaffold is a kind of bone defect repair material with good application prospect.