| Titanium(Ti)-based implants have developed rapidly in recent years and are widely used in clinical treatment of tooth loss.However,in some pathological conditions,such as osteoporotic and diabetic patients,the osseointegration of Ti-based implants may be impaired because of the poor bone quality.Besides,the healing time is too long.Surface-modification of implants for the promotion of early and good osseointegration is a common method to improve the implant bioactivity.The loading of small interfering RNA(siRNA)can realize the surface functionalization of implants,which is a new strategy to improve the osseointegration of implants by specifically targeting and silencing the expression of targeted genes.The delivery system with the abilities of superior siRNA protecting,delivery,and targeting efficiency is needed for the siRNA-biofunctionalized implant to exert its function.In this study,polyethylene glycol(PEG)and polyethylenimine(PEI)dual-functionalized grapheme oxide(GO;nGO-PEG-PEI,GPP)was prepared and optimized for the delivery of siRNA.Firstly,nano-sized nGO-PEG-PEI was prepared,and nGO-PEG-PEI condensed siRNA complex(nGO-PEG-PEI/siRNA)was obtained by optimizing the ratio parameters.Then nGO-PEG-PEI/siRNA was deposited onto the preprepared titanium nanotube(NT)surface via cathodic electrodeposition,obtaining the nGO-PEG-PEI/siRNA-biofunctionalized implant as NT-GPP/siRNA.Casein kinase-2 interacting protein-1(Ckip-1)is a negative regulator of bone formation.After the evaluation of the transfaction and biocompatibility of the implant,siRNA-targeting Ckip-1(siCkip-1)was introduced to the implant,and a series of in vitro and in vivo experiments were carried out to evaluate the osteogenic capacity of NT-GPP/siCkip-1,providing a basis for the functional modification of siRNA on implant surface and guidance for the surface design of functional implants.Part ? Preparation and Characterization of nGO-PEG-PEIObjective:To prepare PEG and PEI dual-functionalized GO(nGO-PEG-PEI)for the delivery of siRNA.Methods:1.Graphene oxide(GO)was prepared by modified Hummers method.2.After alkalization of GO,hydrophilic polymer PEG and positively charged polymer PEI were covalently conjugated to GO with the weight ratio of 1:1:5(GO/PEG/PEI)to obtain nGO-PEG-PEI.3.Characterization of nGO-PEG-PEI:Transmission electron microscopy(TEM)and atomic force microscopy(AFM)were used to observe the morphology of GO,nGO-PEG and nGO-PEG-PEI.Dynamic light scattering(DLS)analyzer was used to measure the particle sizes and surface zeta potential of GO,nGO-PEG and nGO-PEG-PEI.Infrared(IR)spectroscopy was used to observe the functional groups of GO,nGO-PEG and nGO-PEG-PEI,and the conjugation amounts of PEG and PEI werecalculated by elemental analysis and thermogravimetric analysis.Besides,the stability of GO,nGO-PEG and nGO-PEG-PEI in physiological solution was examined.Results:1.GO was successfully prepared by modified Hummers method.The hydrodynamic diameter of GO was about 560 nm,and its thickness was about 3 nm.It was negatively charged,and showed poor solubility in physiological solution.2.The results of IR spectroscopy,elemental analysis and thermogravimetric analysis confirmed that PEG and PEI were successfully conjugated to GO and nGO-PEG-PEI was obtained.The content of N in nGO-PEG-PEI was about 12.84%,and the conjugation amounts of PEG and PEI were 15.7%and 36.9%respectively.3.nGO-PEG-PEI showed unique physical and chemical properties with nanostructure(about 53.9 nm),positively charged surface(about 30.5 mV),and good solubility in water,PBS and?-MEM medium with 10%FBS.Conclusions:GO was successfully prepared by modified Hummers method.Then,PEG and PEI were successfully conjugated to GO to obtain nGO-PEG-PEI,which showed unique physical and chemical properties with nanostructure,positively charged surface,and good solubility in PBS and?-MEM medium with 10%FBS.Part ? Preparation and Characterization of NT-GPP/siRNAObjective:To optimize the N/P ratio and prepare the nGO-PEG-PEI/siRNA biofunctionalized implant.Methods:1.Assigned amounts of nGO-PEG-PEI conjugate were mixed with siRNA at different N/P ratios(5,10,20,40,and 80)to form nGO-PEG-PEI/siRNA complexes.The siRNA binding ability of synthesized nGO-PEG-PEI was evaluated by the agarose gel electrophoresis assay.2.For evaluation of the biocompatibility of nGO-PEG-PEI at different N/P ratios,relative viabilities of MC3T3-E1 cells were determined by the CCK-8 assay after culturing with nGO-PEG-PEI for 1 and 3 days.Besides,the delivery efficiency of nGO-PEG-PEI for siRNA transfection was studied by CLSM and FACS.The gene silencing effect of siRNA transfected cells in each group was further analyzed by PCR,and the final N/P ratio was selected.3.NT samples were first prepared by anodization.Then,the deposition of nGO-PEG-PEI/siRNA on NT samples was carried out in different voltages to form the nGO-PEG-PEI/siRNA-modified NT implants(NT-GPP/siRNA).4.The surface deposition efficiency of siRNA at different voltages was studied,and the appropriate cathodic electrodeposition voltage was screened.Besides,the release characteristics of siRNA from NT-GPP/siRNA implants under this voltage were studied.NT-GPP/siRNA was characterized by AFM,SEM,hydrophilicity measurement and other methods.Results:1.The result of the agarose gel electrophoresis assay showed that nGO-PEG-PEI could completely bind siRNA via electrostatic interaction at N/P ratios above 10.The prepared nGO-PEG-PEI/siRNA complexes showed good biocompatibility among N/P 20,30,and 40 groups.2.The results of CLSM,FACS and PCR showed that the uptake and gene silencing ability of siRNA were significantly increased with the N/P ratio from 20 to 40.The efficiency of gene transfection and gene silencing was the highest in N/P 40 group with target gene silencing up to 70%.3.NT samples were successfully prepared by anodization.nGO-PEG-PEI/siRNA was successfully deposited on NT samples to form NT-GPP/siRNA,and the loading amount is the highest at the voltage of 10 V.4.The release rate of 10 V NT-GPP/siRNA(Cy3)was evaluated,indicating a sustained release of siRNA for 16 days.After the deposition of nGO-PEG-PEI/siRNA,the surface morphology changed significantly and the roughness and hydrophilicity were increased.Conclusions:1.Proper N/P ratio with high siRNA binding capacity,good biocompatibility and high efficiency of gene transfection was selected to prepare nGO-PEG-PEI/siRNA complex.2.nGO-PEG-PEI/siRNA complex was successfully deposited on the surface of NT by cathodic electrodeposition to form NT-GPP/siRNA.3.10 V was chosen for NT-GPP/siRNA preparation with the highest siRNA deposition efficiency.The prepared NT-GPP/siRNA showed stable and sustained release of siRNA,and unique surface morphology and physicochemical properties.Part ? The transfection efficiency of NT-GPP/siRNA in vitroObjective:To evaluate the biocompatibility and the transfection efficiency of siRNA on the implant surface,so as to lay a foundation for the subsequent application in vivo and in vitro.Methods:1.The protein adsorption on NT-GPP/siRNA surface was examined by BCA assay.The morphology and F-actin cytoskeleton distribution of adherent cells on material surface were observed by CLSM and SEM.The proliferation activity of cells was tested by CCK-8 assay.2.The transfection and location of siRNA after transfection was observed by CLSM.GFP~+MC3T3-E1 cells were cultured on NT-GPP/siGFP surface to observe the change of GFP fluorescence expression.The siRNA silencing efficiency of NT-GPP/siCkip-1 was detected by PCR.Results:1.The adsorption capacity of protein on the surface of NT-GPP/siRNA increased significantly,about twice as much as that of PT group and 1.3 times of NT group.2.After cultured on NT-GPP/siRNA,the GFP~+MC3T3-E1 cells extended well,and the filamentous pseudopodia junctions increased.The expression of F-actin in the cytoplasm increased with orderly arrangement.After1,3 and 7 days,NT-GPP/siRNA group could promote cell proliferation without obvious cytotoxicity.3.After transfection of siRNA into cells,siRNA green fluorescence was expressed in the cytoplasm and around the nucleus.After GFP~+MC3T3-E1 cells cultured on the surface of NT-GPP/siGFP,the expression of green fluorescent protein in cells was significantly reduced.Compared with NT-GPP/siNC group,the expression of Ckip-1 of NT-GPP/siCkip-1 group was significantly down-regulated afterboth 3 days and 7 days.Conclusions:1.NT-GPP/siRNA exhibited good biocompatibility,which could promote surface protein adsorption,early cell adhesion and cytoskeleton distribution without obvious cytotoxicity.2.siRNA on NT-GPP/siRNA was efficiently transfected into cells and located in the cytoplasm and around the nucleus.After the RNA interference,the expression of target gene was reduced significantly.Part ? The osteogenesis of NT-GPP/siCkip-1 in vitro and in vivoObjective:To evaluate the in vitro osteogenic differentiation and in vivo osseointegration of NT-GPP/siCkip-1.Methods:1.In vitro osteogenic differentiation:The osteogenic differentiation of MC3T3-E1cells on the NT-GPP/siCkip-1 samples was studied by alkaline phosphatase(ALP)production,collagen secretion,and extracellular matrix(ECM)mineralization staining,and semi-quantitative analysis was performed at setted time points.Besides,the expression of osteogenic related genes ALP,COL-1,BMP2 and Runx2 were measured.2.In vivo osseointegration:NT-GPP/siCkip-1 implants were implanted into the femur of C57BL/6J mice.After one month,in vivo osseointegrationwas evaluated by micro-CT reconstruction analysis,histological VG staining,SEM observation and EDX-ray scanning.Results:1.The collagen secretion and ECM mineralization showed the similar trend with the ALP results,which displayed the following order:NT-GPP/siCkip-1>NT-GPP/siNC?NT-GPP>NT>PT.Additionally,the expression of ALP,COL-1,Runx2 and BMP2 genes in NT-GPP/siCkip-1 group was significantly increased.2 Micro-CT results showed that the amount of new bone formation around NT-GPP/siCkip-1 specimens increased significantly.Histological VG staining showed that NT-GPP/siCkip-1 group generated the highest bone volume and bone continuity than the other groups.SEM and EDX scanning of the bone-to-implant interface showed that the new bone was rich in Ca and P,and the bonding between new bone and the NT-GPP/siCkip-1 implant surface was direct and tight without fibrous or other tissue.Conclusions:1.NT-GPP/siCkip-1 can improve ALP synthesis,collagen secretion and ECM mineralization of osteoblasts,and promote the in vitro osteogenic differentiation.2.NT-GPP/siCkip-1 could significantly improve the in vivo osseointegration.The amount of new bone was the largest,and the bone tissue was compact and continuous,which was tightly contacted with the implant surface. |
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